Laccases, nucleic acids encoding them and methods for making and using them

ABSTRACT

The invention provides laccases, polynucleotides encoding these enzymes, the methods of making and using these polynucleotides and polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.10/567,536, now U.S. Pat. No. 7,741,089, filed Jan. 3, 2007; which is aNational Stage filing of International Application No.PCT/US2004/025932, filed Aug. 11, 2004; which claims the benefit ofpriority to U.S. Application 60/494,472, filed Aug. 11, 2003. All ofthese applications are herein incorporated by reference in theirentirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text as follows:

File Name Date of Creation Size (bytes) D20401D1_SEQLISTING.txt Apr. 21,2010 76.5 KB (78,358 bytes)

FIELD OF THE INVENTION

This invention relates to the fields of biochemistry, and in one aspect,to the enzymatic production of natural flavoring agents for the food andperfume industries. The invention provides laccases, polynucleotidesencoding these enzymes, the use of such polynucleotides andpolypeptides. In one aspect, the invention provides a method for theenzymatic production of nootkatone from valencene using proteins havinga laccase activity, e.g., a novel laccase of the invention. In oneaspect, the nootkatone is produced from valencene using a polypeptidehaving a peroxidase or laccase activity. In one aspect, the inventionprovides methods of depolymerizing lignin, e.g., in a pulp or papermanufacturing process, using a polypeptide of the invention. In anotheraspect, the invention provides methods for oxidizing products that canbe mediators of laccase-catalyzed oxidation reactions, e.g.,2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS),1-hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpiperidin-1-yloxy(TEMPO), dimethoxyphenol, dihydroxyfumaric acid (DHF) and the like.

BACKGROUND

Laccases are a subclass of the multicopper oxidase super family ofenzymes, which includes ascorbate oxidases and the mammalian protein,ceruloplasmin. Laccases are one of the oldest known enzymes and werefirst implicated in the oxidation of urushiol and laccol in the Orientallacquer plant (Rhus vernicifera) by Yoshida in 1883 (Reviewed inMalmström, B. G., “Early and more recent history in the research onmulti-copper oxidases” in Multi-copper oxidases, ed Messercshmidt, A.(1997), World Scientific, Singapore). Work by Bertrand in 1894-7(Malmström, B. G) further characterized the tree laccase as well aslaccases from mushrooms. Laccases are now known to be widespread infungi (Thurston (1994) Microbiology 140:19-26) and also to occur in theentire plant family of the Anacardiaceae (Hutterman (2001) Appl. Microb.Biotechnol. 55:387-394), of which the lacquer plant is a member. Thereare also reports of laccase activities in a variety of other plants (Bao(1993) Science 260:672). Recently there have been several reports ofbacterial enzymes that exhibit laccase activity (Diamantidis, G., et al,(2000), Soil Biology and Biochemistry, 32, 919-927; Sanchez-Amat, A., etal, (1997) A, Biochem. Biophys. Res. Commun., 240, 787-792) and genesencoding putative laccases have been identified in the genomes of manymore bacteria (Alexandre, G., et al, (2000), TIBTECH, 18, 41-42;Solanon, F., et al, (2001), FEMS Microbiol. Lett., 204, 175-81).

The generally accepted reaction catalyzed by laccases is the oxidationof phenolic substrates. In the case of plant laccases this activity isbelieved to result in oligomerization of monolignols in the early stagesof the biosynthesis of lignin (Bao 1993, supra), the most abundantaromatic polymer on earth. In contrast, fungal laccases have beenimplicated in the degradation of lignin—the reversereaction—particularly by white-rot fungi (ten Have (2001) Chem. Rev.101:3397-3413). The major target application has been in thedelignification of wood fibers during the preparation of pulp.

Laccases are found in many plant pathogenic fungi and there are severalreports where laccase production has been correlated with infection(Williamson (1997) Front. Sci., 199, E99-E107). However, there is littleevidence of a clear direct role of the laccase in the plantpathogenesis. In the opportunistic human pathogenic fungus Cryptococcusneoformans (also Filobasidiella noeformans) there is a laccase enzymethat appears to be associated with the pathogenic phenotype. CNLAC1 ispresent in both pathogenic and non-pathogenic species from the genusFilobasidiella (Petter (2001) Microbiology, 147, 2029-2036.), but mayplay a role in protecting the pathogen from attack by the host (Liu(1999) Infect. Immun., 67, 6034-6039). There are no known suchassociations with bacterial laccases.

Laccases catalyze the oxidation of phenolic or other compounds with theconcomitant reduction of oxygen to water (Malmström, 1997, supra). Theycontain four active-site copper ions that mediate electron transferbetween oxidant and reductant (Thurston, 1994, supra, and Petter (2001),Microbiology, 147, 2029-2036). Although the specificity for the electrondonor (substrate) is low, the specificity for the acceptor (oxygen) isabsolute, see FIG. 12A. For example:4-benzenediol+O₂→4-benzosemiquinone+2H₂O

Substrate oxidation by the laccase is a one-electron reaction thatgenerates a free radical from the substrate. This free radical mayundergo one of several reactions: i. further enzyme oxidation to yield,for example, a quinone from phenol; ii. quenching by hydrogenabstraction; or iii. polymerization.

In special cases, oxidation of the substrate yields a stabilized radicalthat can abstract a hydrogen from another organic molecule, therebyreturning to the ground state substrate. In this case, the initialsubstrate is said to act as a mediator and the final product of thereaction is the oxidized form of the second organic compound. Thiscycling of mediator molecules is believed to be a key element oflaccase-catalyzed delignification (ten Have 2001, supra, and Leonowiccz(2001) J. Basic Microbiol. 41:185-227); see FIG. 12B.

A well-studied example of a mediator molecule is 1-hydroxy benzatriazole(HBT) (Fabbrini (2002), J. Mol. Catalysis B: Enzymatic, 16, 231-240),which forms a stable N-oxy radical species when oxidized by a laccase.The oxidized HBT is then able to react with other organic compounds byabstraction of a hydrogen and returning to the reduced state. Thismediator is utilized in the oxidation of valencene to nootkatone; seeFIG. 12C.

The broad substrate specificity of laccases allows their activity to bemeasured by the oxidation of one of several substrates, including2,2′-azinobis(3-ethylbenzthiazoline-sulfonic acid), (ABTS),syringaldizine, and dimethoxyphenol (DMP) (Malstrom 1997, supra,Thurston 1994, supra, and Fabbrini 2002, supra,). In each case theoxidized product absorbs in the visible wavelength range and can beeasily monitored in a spectrophotometer; see FIG. 12D.

The sesquiterpene nootkatone(4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2(3II)-naphtalenone)is an important flavor constituent of grapefruit, which in isolated formis used commercially in perfumery and to flavor soft drinks and otherbeverages. Flavoring agents such as nootkatone are routinely used toenhance product appeal in the food and beverage industry, the cosmeticindustry and the health care industry. The increased demand forflavoring agents in these industries has created a number ofopportunities for biocatalysis (use of enzymes) and fermentation tocompete with traditional synthetic chemistry for the production offlavors.

Current enzymatic methods for the production of nootkatone are limitedin their application due to poor turnover and loss of yield at increasedsubstrate concentrations.

SUMMARY

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQID NO:25 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or moreresidues, encodes at least one polypeptide having a laccase, or aperoxidase, activity, and the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.

In one aspect, the invention provides isolated or recombinant nucleicacids comprising a nucleic acid sequence having at least about 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto SEQ ID NO:1. In one aspect, the invention provides isolated orrecombinant nucleic acids comprising a nucleic acid sequence having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or complete(100%) sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQID NO:17, SEQ ID NO:19 and/or SEQ ID NO:21. In one aspect, the inventionprovides isolated or recombinant nucleic acids comprising a nucleic acidsequence having at least about 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or more, or complete (100%) sequence identity to SEQ ID NO:9. In oneaspect, the invention provides isolated or recombinant nucleic acidscomprising a nucleic acid sequence having at least about 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to SEQ ID NO:13. In one aspect, theinvention provides isolated or recombinant nucleic acids comprising anucleic acid sequence having at least about 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:15.In one aspect, the invention provides isolated or recombinant nucleicacids comprising a nucleic acid sequence having at least about 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more, or complete (100%) sequence identity to SEQID NO:23.

Exemplary nucleic acids of the invention also include isolated orrecombinant nucleic acids encoding a polypeptide having a sequence asset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26 and subsequencesthereof and variants thereof. In one aspect, the polypeptide has alaccase, or a peroxidase, activity.

In one aspect, the invention also provides laccase-encoding nucleicacids with a common novelty in that they are derived from mixedcultures. The invention provides laccase-encoding nucleic acids isolatedfrom mixed cultures comprising a polynucleotide of the invention, e.g.,a sequence having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to an exemplary nucleic acid of the invention, e.g.,SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23 or SEQ ID NO:25 over a region of at least about 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or more.

In one aspect, the invention provides laccase-encoding nucleic acids,and the polypeptides encoded by them, with a common novelty in that theyare derived from a common source, e.g., an environmental or a bacterialsource, e.g., the laccase of SEQ ID NO:6, encoded by the nucleic acid ofSEQ ID NO:5, and the laccase of SEQ ID NO:14, encoded by the nucleicacid of SEQ ID NO:13.

In one aspect, the invention also provides laccase-encoding nucleicacids with a common novelty in that they are derived from environmentalsources, e.g., mixed environmental sources. In one aspect, the inventionprovides laccase-encoding nucleic acids isolated from environmentalsources, e.g., mixed environmental sources, comprising a nucleic acid ofthe invention, e.g., a sequence having at least about 10%, 15%, 20%,25%, 30%, 35%, 40% 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary nucleic acidof the invention over a region of at least about 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200 or more, residues, wherein the nucleicacid encodes at least one polypeptide having a laccase activity, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall -p blastp-d “nr pataa” -F F, and all other options are set to default.

Another aspect of the invention is an isolated or recombinant nucleicacid including at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000,2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or moreconsecutive bases of a nucleic acid sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto.

In one aspect, the laccase activity of the invention comprises thedepolymerization of lignin or the polymerization of lignin. In oneaspect, the laccase activity comprises catalyzing the oxidation ofcommon electron transfer mediators, for example, 1-hydroxybenzotriazole(HBT), N-benzoyl-N-phenyl hydroxylamine (BPHA), N-hydroxyphthalimide,3-hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB),2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), dimethoxyphenol ordihydroxyfumaric acid (DHF).

In one aspect, laccase activity of the polypeptides of the inventioncomprises catalysis of oxidation of dioxygen (O₂) to two molecules ofwater with simultaneously one-electron oxidation of an aromaticsubstrate, e.g., a polyphenol, a methoxy-substituted monophenol, anaromatic amine, or any oxidizable aromatic compound. In one aspect, thelaccase activity of the invention comprises catalysis of oxidization ofa polyphenol, a methoxy-substituted monophenol, an aromatic amine, orany oxidizable aromatic compound.

In one aspect, the laccase activity comprises production of nootkatonefrom valencene. In one aspect, the laccase activity comprises aperoxidase activity. In one aspect, the invention provides a process forthe production of nootkatone comprising formation of a hydroperoxideintermediate, as illustrated in FIG. 5. In one aspect, the hydroperoxideintermediate is converted to nootkatone by heating. In one aspect, theprocess for the production of a nootkatone comprises use of athermotolerant laccase, e.g., a laccase active under conditionscomprising a temperature of at least about 55° C. or greater. In thisaspect, by running the laccase-catalyzed oxidation of valencene at atemperature of at least about 55° C. or greater, the reaction product (ahydroperoxide intermediate) is removed by in situ conversion tonootkatone. In one aspect, the method further comprises conditionscomprising addition of a base, e.g., sodium bicarbonate, to increase pH.Thus, in one aspect, the laccase of the invention, and the laccase usedin the methods of the invention is both thermotolerant and active underalkaline conditions (the laccase of the invention is “alkaliphilic”).

In one aspect, the laccase activity comprises oxidation of a lignin in awood or paper pulp or a wood or paper product. In one aspect, thelaccase activity comprises catalyzing the oxidation of a lignin in afeed, a food product or a beverage. In one aspect, the feed, foodproduct or beverage comprises a cereal-based animal feed, a wort or abeer, a dough, a fruit or a vegetable. In one aspect, the laccaseactivity comprises catalyzing the oxidation of a lignin in a microbialcell, a fungal cell, a mammalian cell or a plant cell.

In one aspect, the laccase activity comprises oxidizing a lignin toproduce a smaller molecular weight polysaccharide or oligomer. In oneaspect, the laccase activity comprises hydrolyzing lignin in cellulose.In one aspect, the laccase activity comprises oxidizing lignin in a woodor paper pulp or a paper product.

In one aspect, the laccase activity comprises catalyzing oxidation oflignins in a cell, e.g., a plant cell or a microbial cell.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a laccase activity that is thermostable. Thepolypeptide can retain a laccase activity under conditions comprising atemperature range of between about 37° C. to about 95° C.; between about55° C. to about 85° C., between about 70° C. to about 95° C., or,between about 90° C. to about 95° C.

In another aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a laccase activity that is thermotolerant. Thepolypeptide can retain a laccase activity after exposure to atemperature in the range from greater than 37° C. to about 95° C. oranywhere in the range from greater than 55° C. to about 85° C. Thepolypeptide can retain a laccase activity after exposure to atemperature in the range between about 1° C. to about 5° C., betweenabout 5° C. to about 15° C., between about 15° C. to about 25° C.,between about 25° C. to about 37° C., between about 37° C. to about 95°C., between about 55° C. to about 85° C., between about 70° C. to about75° C., or between about 90° C. to about 95° C., or more. In one aspect,the polypeptide retains a laccase activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. atabout pH 4.5.

The invention provides isolated or recombinant nucleic acids comprisinga sequence that hybridizes under stringent conditions to a nucleic acidcomprising a sequence of the invention, e.g., a sequence as set forth inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23 or SEQ ID NO:25 or fragments or subsequencesthereof. In one aspect, the nucleic acid encodes a polypeptide having alaccase activity. The nucleic acid can be at least about 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200 or more residues in length or the full length of the gene ortranscript. In one aspect, the stringent conditions include a wash stepcomprising a wash in 0.2×SSC at a temperature of about 65° C. for about15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having a laccase activity, wherein the probecomprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more, consecutivebases of a sequence comprising a sequence of the invention, or fragmentsor subsequences thereof, wherein the probe identifies the nucleic acidby binding or hybridization. The probe can comprise an oligonucleotidecomprising at least about 10 to 50, about 20 to 60, about 30 to 70,about 40 to 80, or about 60 to 100 consecutive bases of a sequencecomprising a sequence of the invention, or fragments or subsequencesthereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having a laccase activity, wherein the probecomprises a nucleic acid comprising a sequence at least about 10, 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residueshaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to a nucleic acid of the invention,wherein the sequence identities are determined by analysis with asequence comparison algorithm or by visual inspection. In alternativeaspects, the probe can comprise an oligonucleotide comprising at leastabout 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about60 to 100 consecutive bases of a nucleic acid sequence of the invention,or a subsequence thereof.

The invention provides an amplification primer pair for amplifying anucleic acid encoding a polypeptide having a laccase activity, whereinthe primer pair is capable of amplifying a nucleic acid comprising asequence of the invention, or fragments or subsequences thereof One oreach member of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50, or more, consecutivebases of the sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive bases of thesequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more residues of anucleic acid of the invention, and a second member having a sequence asset forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 ormore residues of the complementary strand of the first member.

The invention provides laccase-encoding nucleic acids generated byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. The invention provideslaccases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a laccase by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having a laccase activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence of the invention, or fragments orsubsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a cereal, a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a cereal plant, a corn plant, apotato plant, a tomato plant, a wheat plant, an oilseed plant, arapeseed plant, a soybean plant, a rice plant, a barley plant or atobacco plant.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a cereal plant, a corn seed, a wheat kernel,an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed,a sesame seed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a laccase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention. In one aspect, the antisenseoligonucleotide is between about 10 to 50, about 20 to 60, about 30 to70, about 40 to 80, or about 60 to 100 bases in length, e.g., 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ormore bases in length.

The invention provides methods of inhibiting the translation of alaccase message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesdouble-stranded inhibitory RNA (RNAi) molecules comprising a subsequenceof a sequence of the invention. In one aspect, the RNAi is about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more duplexnucleotides in length. The invention provides methods of inhibiting theexpression of a laccase in a cell comprising administering to the cellor expressing in the cell a double-stranded inhibitory RNA (iRNA),wherein the RNA comprises a subsequence of a sequence of the invention.

The invention provides an isolated or recombinant polypeptide comprisingan amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary polypeptide or peptide of the invention over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 ormore residues, or over the full length of the polypeptide, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection. Exemplary polypeptide orpeptide sequences of the invention include SEQ ID N0:2, SEQ ID N0:4, SEQID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID N0:24, SEQ IDN0:26, and subsequences thereof and variants thereof. Exemplarypolypeptides also include fragments of at least about 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600 or more residues in length, or over the fulllength of an enzyme. Exemplary polypeptide or peptide sequences of theinvention include sequence encoded by a nucleic acid of the invention.Exemplary polypeptide or peptide sequences of the invention includepolypeptides or peptides specifically bound by an antibody of theinvention.

In one aspect, a polypeptide of the invention has at least one laccaseactivity. In one aspect, the laccase activity comprises production of anootkatone from a valencene. In one aspect, the laccase activity thelaccase activity comprises an oxidase activity, or, a peroxidaseactivity.

In one aspect, the laccase activity of the invention of a polypeptide ofthe invention comprises catalysis of oxidation of dioxygen (O₂) to twomolecules of water with simultaneously one-electron oxidation of anaromatic substrate, e.g., a polyphenol, a methoxy-substitutedmonophenol, an aromatic amine, or any oxidizable aromatic compound. Inone aspect, the laccase activity of a polypeptide of the inventioncomprises catalysis of oxidization of a polyphenol, amethoxy-substituted monophenol, an aromatic amine, or any oxidizablearomatic compound.

In one aspect, the laccase activity comprises oxidation of lignin in awood or paper pulp or a wood or paper product. In one aspect, thelaccase activity comprises catalyzing the oxidation of a lignin in afeed, a food product or a beverage. In one aspect, the feed, foodproduct or beverage comprises a cereal-based animal feed, a wort or abeer, a dough, a fruit or a vegetable. In one aspect, the laccaseactivity comprises catalyzing the oxidation of a lignin in a microbialcell, a fungal cell, a mammalian cell or a plant cell.

In one aspect, the laccase activity comprises catalyzing oxidation of alignin in a cell, e.g., a plant cell or a microbial cell.

In one aspect, the laccase activity is thermostable. The polypeptide canretain a laccase activity under conditions comprising a temperaturerange of between about 1° C. to about 5° C., between about 5° C. toabout 15° C., between about 15° C. to about 25° C., between about 25° C.to about 37° C., between about 37° C. to about 95° C., between about 55°C. to about 85° C., between about 70° C. to about 75° C., or betweenabout 90° C. to about 95° C., or more. In another aspect, the laccaseactivity can be thermotolerant. The polypeptide can retain a laccaseactivity after exposure to a temperature in the range from greater than37° C. to about 95° C., or in the range from greater than 55° C. toabout 85° C. In one aspect, the polypeptide can retain a laccaseactivity after exposure to a temperature in the range from greater than90° C. to about 95° C. at pH 4.5.

Another aspect of the invention provides an isolated or recombinantpolypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutivebases of a polypeptide or peptide sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto. The peptide can be, e.g., an immunogenic fragment, a motif(e.g., a binding site), a signal sequence, a prepro sequence or anactive site.

The invention provides isolated or recombinant nucleic acids comprisinga sequence encoding a polypeptide having a laccase activity and a signalsequence, wherein the nucleic acid comprises a sequence of theinvention. The signal sequence can be derived from another laccase or anon-laccase (a heterologous) enzyme. The invention provides isolated orrecombinant nucleic acids comprising a sequence encoding a polypeptidehaving a laccase activity, wherein the sequence does not contain asignal sequence and the nucleic acid comprises a sequence of theinvention. In one aspect, the invention provides an isolated orrecombinant polypeptide comprising a polypeptide of the inventionlacking all or part of a signal sequence. In one aspect, the isolated orrecombinant polypeptide can comprise the polypeptide of the inventioncomprising a heterologous signal sequence, such as a heterologouslaccase signal sequence or non-laccase signal sequence.

In one aspect, the invention provides chimeric proteins comprising afirst domain comprising a signal sequence of the invention and at leasta second domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The enzyme can be a laccase.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro sequence and/or acatalytic domain (CD) of the invention and at least a second domaincomprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro sequence and/or catalytic domain (CD). Inone aspect, the heterologous polypeptide or peptide is not a laccase.The heterologous polypeptide or peptide can be amino terminal to,carboxy terminal to or on both ends of the signal peptide (SP), preprosequence and/or catalytic domain (CD).

The invention provides isolated or recombinant nucleic acids encoding achimeric polypeptide, wherein the chimeric polypeptide comprises atleast a first domain comprising signal peptide (SP), a prepro domainand/or a catalytic domain (CD) of the invention and at least a seconddomain comprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro domain and/or catalytic domain (CD).

The invention provides isolated or recombinant signal sequences (e.g.,signal peptides) consisting of or comprising a sequence as set forth inresidues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20,1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28,1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36,1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45,1 to 46 or 1 to 47, of a polypeptide of the invention, e.g., SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26. In one aspect, the invention provides signalsequences comprising the first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino terminalresidues of a polypeptide of the invention. In one aspect, the inventionprovides signal sequences as set forth in Table 1 (e.g., an exemplarysignal sequence of the invention is residues 1 to 38 of SEQ ID NO:6,encoded by the corresponding subsequence of SEQ ID NO:5, etc.):

TABLE 1 SEQ ID NO: Signal (AA) Source 5, 6 1-38 Bacteria 11, 12 1-47Unknown 19, 20 1-25 Unknown 1, 2 None Unknown 15, 16 None Unknown 13, 141-21 Bacteria 7, 8 1-31 or 1-36 Unknown 3, 4 1-37 Unknown  9, 10 1-26Unknown 21, 22 None Unknown 17, 18 None Unknown 23, 24 1-20 Unknown 25,26 None Unknown

In one aspect, the laccase activity comprises a specific activity atabout 37° C. in the range from about 1 to about 1200 units per milligramof protein, or, about 100 to about 1000 units per milligram of protein.In another aspect, the laccase activity comprises a specific activityfrom about 100 to about 1000 units per milligram of protein, or, fromabout 500 to about 750 units per milligram of protein. Alternatively,the laccase activity comprises a specific activity at 37° C. in therange from about 1 to about 750 units per milligram of protein, or, fromabout 500 to about 1200 units per milligram of protein. In one aspect,the laccase activity comprises a specific activity at 37° C. in therange from about 1 to about 500 units per milligram of protein, or, fromabout 750 to about 1000 units per milligram of protein. In anotheraspect, the laccase activity comprises a specific activity at 37° C. inthe range from about 1 to about 250 units per milligram of protein.Alternatively, the laccase activity comprises a specific activity at 37°C. in the range from about 1 to about 100 units per milligram ofprotein.

In another aspect, the thermotolerance comprises retention of at leasthalf of the specific activity of the laccase at 37° C. after beingheated to the elevated temperature. Alternatively, the thermotolerancecan comprise retention of specific activity at 37° C. in the range fromabout 1 to about 1200 units per milligram of protein, or, from about 500to about 1000 units per milligram of protein, after being heated to theelevated temperature. In another aspect, the thermotolerance cancomprise retention of specific activity at 37° C. in the range fromabout 1 to about 500 units per milligram of protein after being heatedto the elevated temperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

In one aspect, the polypeptide can retain laccase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain a laccase activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 11. In one aspect, the polypeptide can retain alaccase activity after exposure to conditions comprising about pH 6.5,pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptidecan retain a laccase activity after exposure to conditions comprisingabout pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH11.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. The second member of theheterodimer can be a different laccase, a different enzyme or anotherprotein. In one aspect, the second domain can be a polypeptide and theheterodimer can be a fusion protein. In one aspect, the second domaincan be an epitope or a tag. In one aspect, the invention provideshomodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having laccase activity,wherein the polypeptide comprises a polypeptide of the invention, apolypeptide encoded by a nucleic acid of the invention, or a polypeptidecomprising a polypeptide of the invention and a second domain. In oneaspect, the polypeptide can be immobilized on a cell, a metal, a resin,a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, abead, a gel, a plate, an array or a capillary tube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention, e.g., an antibody thatspecifically binds to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention.

The invention provides method of isolating or identifying a polypeptidehaving laccase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having a laccase activity.

The invention provides methods of making an anti-laccase antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-laccase antibody. The invention provides methods ofmaking an anti-laccase immune comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate an immuneresponse.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide havinglaccase activity comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing laccase substrate; and (c) contactingthe polypeptide or a fragment or variant thereof of step (a) with thesubstrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having a laccase activity. Inone aspect, the substrate is a lignin, or a small molecule mediator,e.g., 1-hydroxybenzotriazole (HBT), N-benzoyl-N-phenyl hydroxylamine(BPHA), N-hydroxyphthalimide, 3-Hydroxy-1,2,3-benzotriazin-4-one,promazine, 1,8-Dihydroxy-4,5-dinitroanthraquinone, phenoxazine,anthraquinone, 2-hydroxy-1,4-naphthoquinone, phenothiazine,syringaldazine, anthrone, anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB),2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), dimethoxyphenol, and/ordihydroxyfumaric acid (DHF). In one aspect, the substrate comprises anyphenolic compound.

The invention provides methods for identifying laccase substratecomprising the following steps: (a) providing a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as a laccase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of a laccaseactivity comprising the following steps: (a) providing a polypeptide ofthe invention or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a test compound; (c) contacting the polypeptideof step (a) with the test compound of step (b) and measuring an activityof the laccase, wherein a change in the laccase activity measured in thepresence of the test compound compared to the activity in the absence ofthe test compound provides a determination that the test compoundmodulates the laccase activity. In one aspect, the laccase activity canbe measured by providing a laccase substrate (e.g., see above list ofexemplary laccase substrates, e.g., any phenolic compound) and detectinga decrease in the amount of the substrate or an increase in the amountof a reaction product, or, an increase in the amount of the substrate ora decrease in the amount of a reaction product. A decrease in the amountof the substrate or an increase in the amount of the reaction productwith the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an activator of laccase activity. An increase in the amount of thesubstrate or a decrease in the amount of the reaction product with thetest compound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an inhibitorof laccase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a laccase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide having a laccase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having alaccase activity from an environmental sample. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising an amplification primer sequence pair of the invention, e.g.,having at least about 10 to 50 consecutive bases of a sequence of theinvention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a laccase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid of the invention or asubsequence thereof; (b) isolating a nucleic acid from the environmentalsample or treating the environmental sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treatedenvironmental sample of step (b) with the polynucleotide probe of step(a); and (d) isolating a nucleic acid that specifically hybridizes withthe polynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide having a laccase activity from anenvironmental sample. The environmental sample can comprise a watersample, a liquid sample, a soil sample, an air sample or a biologicalsample. In one aspect, the biological sample can be derived from abacterial cell, a protozoan cell, an insect cell, a yeast cell, a plantcell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having a laccase activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid ofthe invention; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid. In one aspect, themethod can further comprise expressing the variant nucleic acid togenerate a variant laccase polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, Gene Site Saturation Mutagenesis™ (GSSM™),synthetic ligation reassembly (SLR) or a combination thereof. In anotheraspect, the modifications, additions or deletions are introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until a laccasehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In one aspect, the variant laccase polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In another aspect, the variant laccase polypeptidehas increased glycosylation as compared to the laccase encoded by atemplate nucleic acid. Alternatively, the variant laccase polypeptidehas a laccase activity under a high temperature, wherein the laccaseencoded by the template nucleic acid is not active under the hightemperature. In one aspect, the method can be iteratively repeated untila laccase coding sequence having an altered codon usage from that of thetemplate nucleic acid is produced. In another aspect, the method can beiteratively repeated until a laccase gene having higher or lower levelof message expression or stability from that of the template nucleicacid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a laccase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a polypeptidehaving a laccase activity; and, (b) identifying a non-preferred or aless preferred codon in the nucleic acid of step (a) and replacing itwith a preferred or neutrally used codon encoding the same amino acid asthe replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a laccase activity; the method comprisingthe following steps: (a) providing a nucleic acid of the invention; and,(b) identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding a laccase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a laccase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a laccasepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having a laccase activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention; and (b) identifying atleast one preferred codon in the nucleic acid of step (a) and replacingit with a non-preferred or less preferred codon encoding the same aminoacid as the replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in a host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified laccase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a nucleic acid of theinvention, and the nucleic acid encodes a laccase active site or alaccase substrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified laccase active sites or substrate binding sites.In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) by a method comprising an optimized directed evolutionsystem, Gene Site Saturation Mutagenesis™ (GSSM™), synthetic ligationreassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, and acombination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises a laccase enzyme encoded by a nucleic acid of theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions. Theinvention provides methods for modifying a small molecule comprising thefollowing steps: (a) providing a laccase enzyme, wherein the enzymecomprises a polypeptide of the invention, or, a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; (b) providing asmall molecule; and (c) reacting the enzyme of step (a) with the smallmolecule of step (b) under conditions that facilitate an enzymaticreaction catalyzed by the laccase enzyme, thereby modifying a smallmolecule by a laccase enzymatic reaction. In one aspect, the method cancomprise a plurality of small molecule substrates for the enzyme of step(a), thereby generating a library of modified small molecules producedby at least one enzymatic reaction catalyzed by the laccase enzyme. Inone aspect, the method can comprise a plurality of additional enzymesunder conditions that facilitate a plurality of biocatalytic reactionsby the enzymes to form a library of modified small molecules produced bythe plurality of enzymatic reactions. In another aspect, the method canfurther comprise the step of testing the library to determine if aparticular modified small molecule that exhibits a desired activity ispresent within the library. The step of testing the library can furthercomprise the steps of systematically eliminating all but one of thebiocatalytic reactions used to produce a portion of the plurality of themodified small molecules within the library by testing the portion ofthe modified small molecule for the presence or absence of theparticular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofa laccase enzyme comprising the steps of: (a) providing a laccaseenzyme, wherein the enzyme comprises a polypeptide of the invention, ora polypeptide encoded by a nucleic acid of the invention, or asubsequence thereof; and (b) deleting a plurality of amino acid residuesfrom the sequence of step (a) and testing the remaining subsequence fora laccase activity, thereby determining a functional fragment of alaccase enzyme. In one aspect, the laccase activity is measured byproviding a laccase substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of a laccase polypeptide, the method comprisingglycosylating a laccase polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe laccase polypeptide. In one aspect, the laccase specific activitycan be thermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant laccasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell. The invention provides methods of expressing aheterologous nucleic acid sequence in a plant cell comprising thefollowing steps: (a) transforming the plant cell with a heterologousnucleic acid sequence operably linked to a promoter, wherein theheterologous nucleic sequence comprises a sequence of the invention; (b)growing the plant under conditions wherein the heterologous nucleicacids sequence is expressed in the plant cell.

The invention provides methods for oxidizing, breaking up or disruptinga lignin-comprising composition comprising the following steps: (a)providing a polypeptide of the invention having a laccase activity, or apolypeptide encoded by a nucleic acid of the invention; (b) providing acomposition comprising a lignin; and (c) contacting the polypeptide ofstep (a) with the composition of step (b) under conditions wherein thelaccase oxidizes, breaks up or disrupts the lignin-comprisingcomposition. In one aspect, the composition comprises a plant cell, abacterial cell, a yeast cell, an insect cell, or an animal cell. Thus,the composition can comprise any plant or plant part, anylignin-containing food or feed, a waste product and the like. Theinvention provides methods for liquefying or removing alignin-comprising composition comprising the following steps: (a)providing a polypeptide of the invention having a laccase activity, or apolypeptide encoded by a nucleic acid of the invention; (b) providing acomposition comprising a lignin; and (c) contacting the polypeptide ofstep (a) with the composition of step (b) under conditions wherein thelaccase removes, softens or liquefies the lignin-comprising composition.

The invention provides detergent compositions comprising a polypeptideof the invention, or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide has a laccase activity. The laccasecan be a nonsurface-active laccase or a surface-active laccase. Thelaccase can be formulated in a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a gelform, a paste or a slurry form. The detergent compositions of theinvention can comprise one or more enzymes in addition to a laccase ofthe invention, such as another laccase, cellulases, hemicellulases,peroxidases, proteases, glucoamylases, amylases, lipases, cutinases,pectinases, reductases, oxidases, phenoloxidases, lipoxygenases,laccases, ligninases, pullulanases, xylanases, tannases, pentosanases,manlanases, β-laccases, arabinosidases, and mixtures thereof. In oneaspect, one, several or all of the enzymes are immobilized by a covalentbinding on an activated polymer, e.g., a polyethylene glycol. In oneaspect, the enzymes are immobilized via a spacer molecule. See, e.g.,U.S. Pat. No. 6,030,933.

The invention provides methods for washing an object comprising thefollowing steps: (a) providing a composition comprising a polypeptide ofthe invention having a laccase activity, or a polypeptide encoded by anucleic acid of the invention; (b) providing an object; and (c)contacting the polypeptide of step (a) and the object of step (b) underconditions wherein the composition can wash the object. The inventionprovides detergent compositions and detergent additives comprising apolypeptide of the invention having a laccase activity, or a polypeptideencoded by a nucleic acid of the invention, and another enzyme, e.g., aprotease, a lipase, an amylase, and/or a cellulase. In one aspect, thelaccase of the invention has stability properties favorable for use witha detergent, e.g., the laccase of the invention used in the detergent isthermostable, is active under alkaline conditions, acid conditions, orboth. See, e.g., U.S. Pat. No. 5,925,554. In one aspect, the laccase ofthe invention has activity profiles favorable for use with a detergent,e.g., the laccase of the invention used in the detergent have anincreased oxidation potential and/or an optimized pH activity optimumand/or an optimized mediator pathway and/or an optimized altered O₂/OH⁻pathway. See, e.g., U.S. Pat. No. 6,060,442. The activity and/orstability properties of the laccase of the invention can be modified bymethods described herein, e.g., by modifying the nucleic acid encodingthe laccase by use of error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, Gene Site Saturation Mutagenesis™ (GSSM™)and/or synthetic ligation reassembly (SLR) or a combination thereof.

The invention provides textiles or fabrics, including, e.g., threads,comprising a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention. In one aspect, the textiles or fabricscomprise lignin-containing fibers. The invention provides methods fortreating a textile or fabric (e.g., removing a stain from a composition)comprising the following steps: (a) providing a composition comprising apolypeptide of the invention having a laccase activity, or a polypeptideencoded by a nucleic acid of the invention; (b) providing a textile orfabric comprising a lignin; and (c) contacting the polypeptide of step(a) and the composition of step (b) under conditions wherein the laccasecan treat the textile or fabric (e.g., remove the stain).

The invention provides dye compositions comprising a polypeptide of theinvention having a laccase activity, or a polypeptide encoded by anucleic acid of the invention. The dye composition of the invention cancomprise a polypeptide of the invention having a laccase activity, or apolypeptide encoded by a nucleic acid of the invention (e.g., apolyporus laccase) and at least one dye precursor capable of beingoxidized by the laccase in the presence of a source of oxygen. See,e.g., U.S. Pat. No. 5,667,531. The invention provides methods for dyingcompositions, e.g., fabrics, using a polypeptide of the invention havinga laccase activity, or a polypeptide encoded by a nucleic acid of theinvention, and at least one dye precursor capable of being oxidized bythe laccase in the presence of a source of oxygen. See, e.g., U.S. Pat.No. 5,667,531. The invention provides processes for providing a bleachedlook in the color density of the surface of dyed fabric, e.g. denim, byusing a phenol-oxidizing laccase of the invention, a hydrogen peroxidesource and an enhancing agent. See, e.g., U.S. Pat. No. 5,752,980. Theinvention provides processes for bleaching dye or colorant in a solutioncomprising use of a laccase of the invention, e.g., a laccase of theinvention having a phenol-oxidizing activity (and, in one aspect,further comprising a second phenol-oxidizing enzyme, e.g., a peroxidase)and an enhancing agent (e.g. acetosyringone). See, e.g., U.S. Pat. No.5,912,405. The invention provides processes for permanent dyeing ofkeratinous fibers, such as hair, fur, hide, and wool, with a dyeingcomposition comprising a laccase of the invention, and, in one aspect,further comprising use of one or more dye precursors and/or modifiers.See, e.g., U.S. Pat. No. 5,948,121. The invention provides a process forremoval of excess dye from newly manufactured printed or dyed fabric oryarn comprising treatment with a rinse liquor comprising use of anenzyme of the invention and, in alternative aspects, a second enzyme,e.g., any enzyme exhibiting a peroxidase activity, an oxidation agent,and/or at least one mediator, e.g., an aliphatic, a cyclo-aliphatic, aheterocyclic or an aromatic compound, which, in one aspect, comprisesthe moiety N—OH, e.g., 1-hydroxybenzotriazole. See, e.g., U.S. Pat. Nos.6,248,134; 6,048,367. The invention provides a process for providing ableached look in the color density of the surface of dyed fabric, e.g.,a cellulosic fabric such as a denim, comprising use of a laccase of theinvention, e.g., a laccase of the invention having a phenol-oxidizingactivity, and, in alternative aspects, a hydrogen peroxide source and/ora phenothiazine or phenoxazine enhancing agent. See, e.g., U.S. Pat. No.5,851,233.

The invention provides methods of oxidizing a substrate in the presenceof a laccase of the invention and an enhancing agent. The inventionprovides methods of oxidizing a substrate comprising use of an enzyme ofthe invention and a second enzyme, e.g., catechol oxidase, monophenolmonooxygenase and/or bilirubin oxidase. The invention provides methodsfor the oxidation of iodide comprising contacting, in an aqueoussolution, a polypeptide of the invention having an oxidase enzymeactivity, e.g., a bilirubin oxidase activity, and a source of ioniciodide (I—), for a time and under conditions sufficient to permit theconversion of ionic iodide to iodine by the enzyme. See, e.g., U.S. Pat.Nos. 5,766,896; 5,885,304. The invention provides methods for enzymaticoxidation of aromatic methyl groups to aldehydes by oxygen, employinglaccase-mediator catalyst and the diammonium salt comprising use of anenzyme of the invention. See, e.g., U.S. Pat. No. 5,888,787.

The invention provides methods of bleaching dye in solutions using alaccase of the invention. The invention provides methods of inhibitingthe transfer of a textile dye from a dyed fabric to another fabric whenthe fabrics are washed together in a wash liquor using a laccase of theinvention. See, e.g., U.S. Pat. No. 5,795,855. The invention providesdye compositions comprising a polypeptide of the invention having alaccase activity, or a polypeptide encoded by a nucleic acid of theinvention and at least one dye precursor capable of being oxidized bythe laccase in the presence of a source of oxygen. See, e.g., U.S. Pat.No. 5,981,243.

The invention provides methods of decolorizing a dyed material in apredetermined pattern by providing a dyed material and ink jet printinga solution of a laccase of the invention, e.g., a polypeptide of theinvention having oxidoreductase activity, onto the dyed material in apredetermined pattern. In one aspect, the method comprises a dyedmaterial that has been decolorized in a predetermined pattern by thesemethods. The invention provides methods of simultaneously decolorizingand printing on a dyed material in a predetermined pattern by providinga dyed material and ink jet printing on the dyed material in apredetermined pattern with an ink jet ink comprising a laccase of theinvention, e.g., a polypeptide of the invention having oxidoreductaseactivity. In one aspect, the method can further comprise one or moredyes, which, in one aspect, are not significantly decolorized by thelaccase of the invention. In one aspect, the dyed material is heatedafter printing. See, e.g., U.S. Pat. No. 6,322,596.

The invention provides feeds or foods comprising a polypeptide of theinvention, or a polypeptide encoded by a nucleic acid of the invention.In one aspect, the invention provides a food, feed, a liquid, e.g., abeverage (such as a fruit juice or a beer), a bread or a dough or abread product, or a beverage precursor (e.g., a wort), comprising apolypeptide of the invention. The beverage or a beverage precursor canbe a fruit juice, a beer or a wort. In one aspect, the inventionprovides methods for the clarification of a liquid, e.g., a juice, suchas a fruit juice, or a beer, by treating the liquid with an enzyme ofthe invention. In one aspect, the invention provides methods of doughconditioning comprising contacting a dough or a bread product with atleast one polypeptide of the invention under conditions sufficient forconditioning the dough. See, e.g., U.S. Pat. No. 6,296,883. In oneaspect, the invention provides methods of beverage production comprisingadministration of at least one polypeptide of the invention to abeverage or a beverage precursor under conditions sufficient fordecreasing the viscosity of the beverage.

The invention provides methods for oxidizing a lignin in a feed or afood prior to consumption by an animal comprising the following steps:(a) obtaining a feed material comprising a laccase of the invention, ora laccase encoded by a nucleic acid of the invention; and (b) adding thepolypeptide of step (a) to the feed or food material in an amountsufficient for a sufficient time period to cause oxidation of a ligninand formation of a treated food or feed, thereby oxidizing a lignin inthe food or the feed prior to consumption by the animal. In one aspect,the invention provides methods for oxidizing a lignin in a feed or afood after consumption by an animal comprising the following steps: (a)obtaining a feed material comprising a laccase of the invention, or alaccase encoded by a nucleic acid of the invention; (b) adding thepolypeptide of step (a) to the feed or food material; and (c)administering the feed or food material to the animal, wherein afterconsumption, the laccase causes oxidation of a lignin in the feed orfood in the digestive tract of the animal. The food or the feed can be,e.g., a cereal, a grain, a corn and the like.

The invention provides processes for preparing cork articles, inparticular cork stoppers for wine bottles, which involves treating corkwith a laccase of the invention, or a laccase encoded by a nucleic acidof the invention, e.g., a polypeptide of the invention having a phenoloxidizing enzyme activity, thereby reducing the characteristic corktaint and/or astringency which is frequently imparted to a bottled wineby an untreated cork. The invention provides a cork article, e.g., acork stopper, comprising a laccase of the invention, or a laccaseencoded by a nucleic acid of the invention, e.g., a polypeptide of theinvention having a phenol oxidizing enzyme activity. See, e.g., U.S.Pat. No. 6,152,966.

In another aspect, the invention provides methods for decreasing theviscosity of cellulose in a composition, e.g., in a food or a feed, bytreating the composition with a laccase of the invention, or, includinga laccase of the invention in the composition.

The invention provides food or nutritional supplements for an animalcomprising a polypeptide of the invention, e.g., a polypeptide encodedby the nucleic acid of the invention. In one aspect, the polypeptide inthe food or nutritional supplement can be glycosylated. The inventionprovides edible enzyme delivery matrices comprising a polypeptide of theinvention, e.g., a polypeptide encoded by the nucleic acid of theinvention. In one aspect, the delivery matrix comprises a pellet. In oneaspect, the polypeptide can be glycosylated. In one aspect, the laccaseactivity is thermotolerant. In another aspect, the laccase activity isthermostable.

The invention provides a food, a feed or a nutritional supplementcomprising a polypeptide of the invention. The invention providesmethods for utilizing a laccase as a nutritional supplement in an animaldiet, the method comprising: preparing a nutritional supplementcontaining a laccase enzyme comprising at least thirty contiguous aminoacids of a polypeptide of the invention; and administering thenutritional supplement to an animal to increase utilization of a glucancontained in a feed or a food ingested by the animal. The animal can bea human, a ruminant or a monogastric animal. The laccase enzyme can beprepared by expression of a polynucleotide encoding the laccase in anorganism selected from the group consisting of a bacterium, a yeast, aplant, an insect, a fungus and an animal. The organism can be selectedfrom the group consisting of an S. pombe, S. cerevisiae, Pichiapastoris, E. coli, Streptomyces sp., Bacillus sp. and Lactobacillus sp.

The invention provides edible enzyme delivery matrix comprising athermostable recombinant laccase enzyme, e.g., a polypeptide of theinvention. The invention provides methods for delivering a laccasesupplement to an animal, the method comprising: preparing an edibleenzyme delivery matrix in the form of pellets comprising a granulateedible carrier and a thermostable recombinant laccase enzyme, whereinthe pellets readily disperse the laccase enzyme contained therein intoaqueous media, and administering the edible enzyme delivery matrix tothe animal. The recombinant laccase enzyme can comprise a polypeptide ofthe invention. The laccase enzyme can be glycosylated to providethermostability at pelletizing conditions. The delivery matrix can beformed by pelletizing a mixture comprising a grain germ and a laccase.The pelletizing conditions can include application of steam. Thepelletizing conditions can comprise application of a temperature inexcess of about 80° C. for about 5 minutes and the enzyme retains aspecific activity of at least 350 to about 900 units per milligram ofenzyme.

The laccases of the invention are used to break down the high molecularweight lignins in animal feed. Adding laccases of the inventionstimulates growth rates by improving digestibility, which also improvesthe quality of the animal litter. The laccase of the invention functionsthrough the gastro-intestinal tract to reduce intestinal viscosity andincrease diffusion of pancreatic enzymes. Additionally, the laccases ofthe invention may be used in the treatment of endosperm cell walls offeed grains and vegetable proteins. In one aspect of the invention, thenovel laccases of the invention are administered to an animal in orderto increase the utilization of a lignin in the food. This activity ofthe laccases of the invention may be used to break down insoluble cellwall material, liberating nutrients in the cell walls, which then becomeavailable to the animal. A laccase can also produce a compound that maybe a nutritive source for a ruminal microflora.

The invention provides methods of deoxygenation of an oil or anoil-containing product (e.g., a salad dressing), by adding an effectiveamount of a laccase of the invention. In one aspect, the substrate forthe laccase comprises a mustard, a paprika or a lemon juice. See, e.g.,U.S. Pat. No. 5,980,956. The invention provides methods of gelling apectic material, e.g., a material from a member of the plant familyChenopodiaceae, (e.g., sugar beet) using a laccase of the invention and,in one aspect, a second enzyme, e.g., a pectinesterase and. Theinvention provides methods treating an aqueous medium or a gellablepolymeric material with an effective amount of a laccase of theinvention to cause gelling or increase the viscosity of a gellablepolymeric material. In one aspect, the material comprises a materialhaving a phenolic hydroxy group, an arabinoxylan-extracted from flour orbran and/or a pectin-extraction from member of the familyChenopodiaceae, e.g. sugar beets. See, e.g., U.S. Pat. No. 6,232,101.

The invention provides methods for enhancing flavors or colors in a foodor a feed, including solids or liquids, using a laccase of theinvention. For example, the invention provides methods for enhancingcolor in tea-based products treated with a laccase of the invention. Inone aspect, laccase has a polyphenol oxidase or peroxidase activity. Inone aspect, the laccase is used in combination with, or, with apretreatment, with a tannase. See, e.g., U.S. Pat. No. 5,879,730.

The invention provides a tobacco product (e.g., a cigarette, a cigar,pipe tobacco, a chewing tobacco) comprising a laccase of the invention.The invention provides tobacco products comprising a laccase of theinvention having a reduced amount of phenolic compounds. The inventionprovides tobacco products having a reduced amount of phenolic compounds,wherein they have been treated with a laccase of the invention, but allor most of the laccase of the invention has been removed and/orinactivated. The invention provides processes for preparing tobaccousing a laccase of the invention. In one aspect, the process comprisesthe steps of treating a tobacco material with a laccase of theinvention, e.g., a laccase of the invention having a phenol oxidizingactivity. In one aspect, the process can comprise extracting tobaccowith a solvent to provide an extract and a residue and treating theextract with a laccase of the invention having a phenol oxidizingactivity. In alternative aspects, the process can comprise further stepsof removing the oxidized phenolic compound, adding adsorbents such asbentonite; removing and/or inactivating the enzyme; and/or concentratingthe extract. The treated extract can be re-combined with a tobaccoresidue. The treated extract can be further processed to provide atobacco article for smoking See, e.g., U.S. Pat. No. 6,298,859.

The invention provides methods to increase viscosity of an aqueousmedium, treating it with a laccase of the invention and an oxidizingagent. In one aspect, the method comprises using a second enzyme, e.g.,a carboxylic ester hydrolase, an oxidase and an oxidizing agent. See,e.g., U.S. Pat. No. 5,998,176.

The invention provides papers or paper products or paper pulp comprisinga laccase of the invention, or a polypeptide encoded by a nucleic acidof the invention. The invention provides pulp bleaching processes usinga laccase, e.g., a laccase of the invention, with or without theaddition of a mediator. In processes where no mediator is used,lignosulfonate is used as a “mediator” in a pulp bleaching processwithout the addition of another small molecule, as discussed in Example1, below.

The invention provides methods for treating a paper or a paper or woodpulp comprising the following steps: (a) providing a polypeptide of theinvention having a laccase activity, or a laccase encoded by a nucleicacid of the invention; (b) providing a composition comprising a paper ora paper or wood pulp; and (c) contacting the polypeptide of step (a) andthe composition of step (b) under conditions wherein the laccase cantreat the paper or paper or wood pulp. The invention provides methods ofbleaching a lignin-containing material, and in one aspect, bleaching ofpulp for paper production, using a laccase of the invention. Theinvention provides methods of treatment of waste water from pulp orpaper manufacturing using a laccase of the invention. See, e.g., U.S.Pat. No. 5,795,855.

The invention provides methods for deinking and/or decolorizing a paperor paper product, e.g., a printed paper comprising use of a laccase ofthe invention, or a polypeptide encoded by a nucleic acid of theinvention. In one aspect, the methods comprise pulping a paper, e.g., aprinted paper, to obtain a pulp slurry, dislodging an ink from the pulpslurry with a laccase of the invention, and, in one aspect, one or moreadditional enzymes. In one aspect, the methods further comprisedecolorizing the dye contained in the pulp slurry with a laccase of theinvention in the presence of oxygen. In one aspect, the methods furthercomprise use of one or more chemical mediators, e.g., methyl syringate.In one aspect, the methods further comprise separating the released inkfrom the pulp slurry. In one aspect, the methods further compriserecovering the decolorized pulp. In one aspect, the methods furthercomprise decolorizing pulps for producing recycled paper. See, e.g.,U.S. Pat. No. 6,241,849.

The invention provides methods and apparatus for monitoring andcontrolling a characteristic of process waters or effluents, e.g., fromwood pulp bleaching, pulping and paper making processes utilizing alaccase of the invention, and, in alternative aspects, furthercomprising other laccases and/or a bleaching agent, e.g., hydrogenperoxide (H₂O₂), Na₂, S₂, O₂, ClO₂, Cl₂ and/or O₃. The inventionprovides methods and apparatus for pulp delignification utilizing alaccase of the invention, and, in alternative aspects, otherdelignification agents, such as, e.g., NaOH, Na₂, S, O₂, Na₂, SO₃,and/or enzymes such as ligninase, xylanase, mannanase, other laccasesand/or peroxidase. In one aspect, the methods comprise obtaining several(e.g., at least three) measurements of ultraviolet-visible light fromthe effluent. In one aspect, the measurements comprise taking a firstmeasurement measured at a first wavelength, a second measurementmeasured at a second wavelength, and a third measurement at a thirdwavelength, formulating two ratios from the three measurements and usingthe ratios for computing an empirical value of the characteristic of theeffluent. Feedback control can be used for adjusting feed inputcomponents in accordance with the computed empirical value of thecharacteristic such that a target measurement of the characteristic isobtained. See, e.g., U.S. Pat. No. 6,023,065.

In one aspect, invention provides a pharmaceutical compositioncomprising a laccase of the invention, or a polypeptide encoded by anucleic acid of the invention. In one aspect, the pharmaceuticalcomposition acts as a digestive aid. In one aspect, the pharmaceuticalcomposition is used for oxidation of both conjugated and unconjugatedbilirubin to biliverdin without the formation of hydrogen peroxide;thus, the pharmaceutical composition (the laccase of the invention) isused to prevent the production of hydrogen peroxide. In one aspect, thetreatment is prophylactic. See, e.g., U.S. Pat. No. 4,554,249.

In one aspect, the pharmaceutical composition is used in the treatmentand/or prevention of a dermatitis, e.g., poison ivy dermatitis. In oneaspect, the laccase used in the pharmaceutical composition has anoxidase, e.g., a para-diphenol oxidase, activity. Thus, in one aspect,the pharmaceutical composition of the invention is formulated as atopical formulation, e.g., a lotion or a cream or a spray. In oneaspect, invention provides methods for the treatment and/or preventionof a dermatitis, e.g., a poison ivy dermatitis using a laccase of theinvention, e.g., a laccase having an oxidase, e.g., a para-diphenoloxidase, activity. In one aspect, the methods of the invention comprisetopical application of the pharmaceutical composition to a skin surfacebefore or after exposure to an agent, e.g., an irritant, e.g., a poisonivy irritant, such as urushiol. See, e.g., U.S. Pat. No. 4,259,318.

In one aspect, invention provides methods of killing and inhibiting thegrowth of microorganisms in industrial processes. In one aspect, themethods comprise industrial process streams comprising the addition ofan enzymatically catalyzed biocide system utilizing a laccase of theinvention, e.g., a laccase having an oxidase or a peroxidase activity.In one aspect, the method comprises use of a laccase of the invention inthe presence of an oxidant, e.g., hydrogen peroxide or oxygen to oxidizehalide salts, and/or a phenolic compound. The laccases of the inventioncan be formulated such that they can be added to a process stream toproduce oxidation products that are toxic to microorganisms. See, e.g.,U.S. Pat. No. 4,370,199.

In one aspect, invention provides a cleaning or a disinfectingcomposition comprising a laccase of the invention, or a polypeptideencoded by a nucleic acid of the invention. In one aspect, the inventionprovides methods for cleaning and/or disinfecting a surface, e.g., abiofilm surface, by a cleaning composition of the invention. Thecleaning or disinfecting composition of the invention can furthercomprise a hydrolase, an oxidoreductase, an oxidase, a peroxidase and/oran oxidation enhancer, such as methyl syringate. The surface cancomprise a medical device or instrument, a medical implant or catheter,a surgical device, a dressing and the like. See, e.g., U.S. Pat. No.6,100,080. In one aspect, the invention provides methods foranti-microbial treatment of a composition or liquid, e.g., a surfacecomprising use of a laccase of the invention, or a polypeptide encodedby a nucleic acid of the invention. In one aspect, the inventionprovides methods for treating (e.g., reducing or eliminating)microorganisms and/or viruses on a surface. In one aspect, the methodsfurther comprise use of one or more enhancers in the presence of oxygen.The processes of the invention can be used, e.g., on the surface of ahospital room or surgery, a room for processing food or water treatment,a laboratory and/or a room for chemical or pharmaceutical processing.See, e.g., U.S. Pat. No. 6,228,128.

In one aspect, invention provides methods for reducing oxygen gas in aconfined space or compartment using a laccase of the invention, or apolypeptide encoded by a nucleic acid of the invention. In one aspect,invention provides methods for colorimetrically detecting, orindicating, the presence of an oxygen gas in a confined space orcompartment using a laccase of the invention, or a polypeptide encodedby a nucleic acid of the invention. See, e.g., U.S. Pat. No. 5,654,164.

In one aspect, invention provides methods for cross-linking a protein,using a polypeptide of the invention having an oxidase activity, e.g., amulti-copper oxidase activity, and, in one aspect, a bilirubin oxidase,an ascorbic acid oxidase and/or a ceruloplasmin. See, e.g., U.S. Pat.No. 6,121,013.

In one aspect, the invention provides methods of depolymerizing lignin,e.g., in a pulp or paper manufacturing process, using a polypeptide ofthe invention. In one aspect, the polypeptide of the invention has alaccase activity under alkaline processing conditions, e.g., pH 8, 9, 10or more.

In another aspect, the invention provides methods for oxidizing productsthat can be mediators of laccase-catalyzed oxidation reactions, e.g.,2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS),1-hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpiperidin-1-yloxy(TEMPO), dimethoxyphenol, dihydroxyfumaric acid (DHF) and the like.

The invention provides methods for the enzymatic production ofnootkatones from valencene using proteins having a laccase activity,e.g., a novel laccase of the invention. In one aspect, the nootkatonecomprises a(−)-(4S,4aR,6S)-6-isopropenyl-4,4a-dimethyl-4,4a,5,6,7,8-hexahydro-3H-naphthalen-2-one(i.e., (−)-(4S,4aR,6S)-nootkatone), or, a(+)-(4R,4aS,6R)-6-isopropenyl-4,4a-dimethyl-4,4a,5,6,7,8-hexahydro-3H-naphthalen-2-one(i.e., (+)-(4R,4aS,6R)-nootkatone), or, equivalent compounds. In somesituations, nootkatone can be more efficiently produced enzymaticallywith thermostable (i.e., thermophilic) laccase polypeptides.Accordingly, in one aspect, the invention provides thermostable (i.e.,thermophilic) laccases. In one aspect, the invention provides methodsfor producing nootkatone by utilizing enzyme catalyzed reactions atelevated temperatures using thermophilic laccase polypeptides, e.g., thethermostable (i.e., thermophilic) laccases of the invention.

In one aspect, the invention provides a method for producing nootkatone.The method comprises reacting valencene at a concentration of at least0.1% (v/v) and a composition having laccase activity at temperatureselected from the range of about 4° C. to 75° C., in the presence of anoxygen source and recovering nootkatone from the reaction.

In one aspect, a valencene and a polypeptide of the invention having alaccase activity are reacted in the presence of a catalyst. In someaspects, the catalyst is iron, ascorbic acid, cobalt and/or copper andcombinations thereof. In other aspects of the invention, valencene and apolypeptide of the invention having a laccase activity are reacted inthe presence of a mediator. In some aspects, the mediator is selectedfrom the group consisting of 1-hydroxybenzotriazole (HBT),N-benzoyl-N-phenyl hydroxylamine (BPHA), N-hydroxyphthalimide,3-Hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-Dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV) and/or 2,3-dihydroxybenzoic acid (2,3-DHB) and combinationsthereof.

In certain other aspects, valencene and a polypeptide of the inventionhaving a laccase activity are reacted at a temperature of 55° C., 60°C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or more. In otheraspects, the reaction is done at a pH selected from pH 3.0 to 10.0, ormore, e.g., pH 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. In some aspects, theoxygen source for the reaction is a mixed gas or pure oxygen. In otheraspects, a polypeptide having a laccase activity used in these (or any)method of the invention is a laccase of the invention, e.g., apolypeptide encoded by a nucleic acid having 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQID NO:25, or, to an exemplary polypeptide of the invention, e.g., SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24 or SEQ ID NO:26.

In another aspect, the invention provides a method of producingnootkatone by contacting valencene with a polypeptide of the inventionhaving a laccase activity, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20 or SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, toproduce nootkatone, and recovering the produced nootkatone. In certainaspects, valencene is contacted by the protein at a temperature in therange of between about 4° C. to 75° C., 80° C., 85° C., 90° C., or more.

In some aspects, valencene and a polypeptide of the invention having alaccase activity are reacted in the presence of a catalyst. In certainaspects thereof, the catalyst is iron, ascorbic acid, cobalt and/orcopper and combinations thereof. In other aspects of the invention,valencene and a polypeptide of the invention having a laccase activityare reacted in the presence of a mediator. In some aspects, the mediatoris 1-hydroxybenzotriazole (HBT), N-benzoyl-N-phenyl hydroxylamine(BPHA), N-hydroxyphthalimide, 3-Hydroxy-1,2,3-benzotriazin-4-one,promazine, 1,8-Dihydroxy-4,5-dinitroanthraquinone, phenoxazine,anthraquinone, 2-hydroxy-1,4-naphthoquinone, phenothiazine,syringaldazine, anthrone, anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV) and/or 2,3-dihydroxybenzoic acid (2,3-DHB) and combinationsthereof.

In certain aspects of this aspect, valencene is contacted a polypeptideof the invention having a laccase activity at a pH in the range ofbetween about pH 3.0 to 9.0, 10.0, 11.0 or more. In other aspects,valencene is present at a concentration of at least 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% (v/v) or more when contactedwith the laccase. In other aspects, valencene is contacted with thelaccase activity at a temperature of about 55° C., 60° C., 65° C., 70°C., 75° C., 80° C., 85° C., 90° C., or more.

In another aspect, the invention provides a composition comprisingnootkatone made by the methods described herein.

The details of one or more aspects of the invention are set forth in theaccompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings are illustrative of aspects of the invention andare not meant to limit the scope of the invention as encompassed by theclaims.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 is illustration of an exemplary process of the invention whereina polypeptide of the invention having a laccase activity catalyzes, witha mediator, the conversion of valencene to nootkatone.

FIG. 6 is a graphic illustration of the results of an exemplary assay totest for laccase activity, as described in detail in Example 1, below.

FIG. 7 summarizes data from experiments demonstrating thatlignosulfonate can be used as a “mediator” in a pulp bleaching processof the invention without the addition of another small molecule, asdescribed in detail in Example 1, below.

FIG. 8 illustrates a table summarizing data from tests demonstrating theability of laccases of the invention to oxidize the mediators ABTS, HBTand TEMPO, and lignin, as described in detail in Example 1, below.

FIG. 9 graphically summarizes data from the tests of thelignin-oxidizing activity of an exemplary laccase of the inventionhaving a sequence as set forth in SEQ ID NO:16 (encoded by SEQ ID NO:15)under three different temperatures was also tested, as described indetail in Example 1, below.

FIG. 10 and FIG. 11 illustrate alignments of exemplary sequences of theinvention to illustrate shared structural elements of laccases of theinvention, as discussed in detail, below.

FIGS. 12A, 12B, and 12C illustrate exemplary reactions that can uselaccases of the invention, and FIG. 12D illustrates exemplary substratesand products of exemplary reactions using enzymes of the invention, asdiscussed herein.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides novel laccases, polynucleotides encoding theseenzymes, the use of such polynucleotides and polypeptides. In oneaspect, the laccases of the invention have different, e.g., improved,qualities over known laccases.

The invention provides methods for the enzymatic conversion of valenceneto nootkatone by treatment with a laccase polypeptide of the invention.In certain aspects, the invention provides advantages over the priormethods for the production of nootkatone by utilizing laccasepolypeptides that function at elevated temperatures (e.g., greater than4° C.) and/or under alkaline conditions. Laccase enzymes with one ormore of these characteristics allow for reaction conditions thatfacilitate the conversion of the hydroperoxide intermediate tonootkatone. Prior enzymatic processes for the production of nootkatonefrom valencene required post-enzymatic treatment to convert thehydroperoxide to the end product.

In certain aspects, the substrate utilized in the methods of theinvention is valencene (5,6 dimethyl-8-isopropenyl bicyclo[4.4.0]-1-decene), a compound naturally found in citrus fruit.Commercial sources of valencene are readily available, e.g., GivaudanRoure Flavors, Lakeland, Fla. Starting concentrations of valencene inthese exemplary methods of the invention range from about 0.1% to 50%.In certain aspects, valencene is present at a concentration of at least0.1% or at least 1.0% or at least 10% or at least 25% or at least 50%.Exemplary reaction conditions for the methods of the invention providefor contacting valencene with a laccase polypeptide of the invention,e.g., a thermophilic and/or alkaphilic laccase polypeptide, in asuitable container in the presence of an abundant supply of oxygen.Oxygen can be supplied to the reaction as either a mixed gas containingoxygen, e.g., air, or in pure form.

In certain aspects, the enzymatic conversion of valencene to nootkatoneby the methods of the invention can be done at any temperature, e.g.,20° C. to 75° C. In certain aspects, the temperature of the reaction isa temperature greater than or equal to 4° C. In some aspects, thetemperature of the reaction is about 55° C. In certain aspects,depending on the amount of starting material, methods of the inventioncan employ reaction times varying from as little as 1 hour to one ormore days or weeks or more.

The pH of reaction conditions utilized by the invention is anothervariable parameter for which the invention provides. In certain aspects,the pH of the reaction is conducted in the range of about 3.0 to about9.0. In other aspects, the pH is about 4.5 or the pH is about 7.5 or thepH is about 9. Reaction conditions conducted under alkaline conditionsare particularly advantageous, as basic conditions promote theconversion of the hydroperoxide intermediate to nootkatone.

In certain aspects, the methods of the invention provide for reactionconditions that include catalysts and/or mediators. Exemplary catalystscan be present at a concentration of 1 μM to 10 mM and include, e.g.,iron, or ascorbic acid, or cobalt, or copper or combinations thesecatalysts. In certain aspects, mediators are used; they can beparticularly useful for inclusion in the reaction conditions, e.g., atconcentrations ranging from 0 to 100 mM. In certain aspects, exemplarymediators comprise 1-hydroxybenzotriazole (HBT), N-benzoyl-N-phenylhydroxylamine (BPHA), N-hydroxyphthalimide,3-Hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-Dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthrarufin, anthrarobin, or2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ordimethoxyphenol (DMP), or ferulic acid, or catechin, or epicatechin, orhomovanillic acid (HMV), or 2,3-dihydroxybenzoic acid (2,3-DHB) orcombinations of these mediators.

In another aspect, the reaction intermediate valencene hydroperoxideaccumulates to levels up to 60% of valencene or 30% (v/v) in thereaction mixture. In certain aspects thereof, the valencenehydroperoxide is converted to nootkatone and/or nootkatol in thepresence of a catalyst or protein such as horse-radish peroxidase,lactoperoxidase, chloroperoxidase, lignin peroxidase, soybean peroxidaseor manganese peroxidase, or combinations thereof. In some aspects, thecatalyst or protein is present at all times in the reaction mixture. Inone aspect, the protein or catalyst can be added at an amount anywherein the range of between about 1 unit/mL to 10,000 units/mL. In otheraspects, the catalyst or protein is added once the laccase-catalyzedreaction is complete and is added at an amount anywhere in the range ofbetween about 1 unit/mL to 10,000 units/mL. In another aspect, thevalencene hydroperoxide is converted to nootkatone and/or nootkatol inthe presence of ascorbic acid. In one aspect, the catalyst is added oncethe laccase-catalyzed reaction is complete and is added atconcentrations of an amount anywhere in the range of between about 1 mMto 100 mM.

The invention provides for laccase polypeptides of the invention in avariety of forms and formulations. In the methods of the invention,laccase polypeptides of the invention are used in a variety of forms andformulations. For example, purified laccase polypeptides can be utilizedto contact valencene for the conversion to nootkatone. Alternatively,the laccase polypeptide can be expressed in a microorganism usingprocedures known in the art. In other aspects, the laccase polypeptidesof the invention can be immobilized on a solid support prior to use inthe methods of the invention. Methods for immobilizing enzymes on solidsupports are commonly known in the art, for example J. Mol. Cat. B:Enzymatic 6 (1999) 29-39; Chivata et al. Biocatalysis: Immobilized cellsand enzymes, J Mol. Cat. 37 (1986) 1-24: Sharma et al., ImmobilizedBiomaterials Techniques and Applications, Angew. Chem. Int. Ed. Engl. 21(1982) 837-54: Laskin (Ed.), Enzymes and Immobilized Cells inBiotechnology. As will be understood in the art, the immobilization oflaccase polypeptides of the invention enables higher startingconcentrations of valencene to be utilized in the methods of theinvention, e.g., 50% or more.

The laccase molecules of the instant invention are novel with respect totheir structures and with respect to their origin. Additionally, theinstant laccase molecules have novel activity at elevated temperaturesand/or under alkaline conditions.

Definitions

As used herein, the term “laccase” encompasses any polypeptide orenzymes having any laccase activity, for example, enzymes capable ofcatalyzing the conversion of valencene to nootkatone, as illustrated inFIG. 5. In one aspect, the laccase activity comprises catalyzing theoxidation of lignin. In one aspect, the laccase activity comprises thedepolymerization or polymerization of lignin. In one aspect, the laccaseactivity comprises catalyzing the oxidation of 1-hydroxybenzotriazole(HBT), N-benzoyl-N-phenyl hydroxylamine (BPHA), N-hydroxyphthalimide,3-hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB),2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), dimethoxyphenol ordihydroxyfumaric acid (DHF) or equivalent compounds.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense (complementary) strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material, natural or synthetic inorigin. The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double strandediRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. “Oligonucleotide” includeseither a single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides have no 5′ phosphate and thus will not ligateto another oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide can ligate to afragment that has not been dephosphorylated.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which is transcribedand translated into a polypeptide or protein when placed under thecontrol of appropriate regulatory sequences.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons). “Operably linked” as used herein refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments.Typically, it refers to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid of theinvention, if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as a laccase of the invention) ina host compatible with such sequences. Expression cassettes include atleast a promoter operably linked with the polypeptide coding sequence;and, optionally, with other sequences, e.g., transcription terminationsignals. Additional factors necessary or helpful in effecting expressionmay also be used, e.g., enhancers, alpha-factors. Thus, expressioncassettes also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like. A“vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these and to naturally occurringor synthetic molecules.

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterm “polypeptide” as used herein, refers to amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres and may contain modified amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Modificationscan occur anywhere in the polypeptide, including the peptide backbone,the amino acid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also agiven polypeptide may have many types of modifications. Modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment of aphosphytidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, pegylation, glucanhydrolase processing, phosphorylation, prenylation, racemization,selenoylation, sulfation and transfer-RNA mediated addition of aminoacids to protein such as arginylation. (See Creighton, T. E.,Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)). The peptides and polypeptides of the invention also include all“mimetic” and “peptidomimetic” forms, as described in further detail,below.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. However, the term “purified” also includes nucleicacids which have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders and more typicallyfour or five orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. Additionally, to be “enriched” the nucleic acidswill represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid backbone molecules. Backbone moleculesaccording to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. Typically, the enriched nucleic acidsrepresent 15% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. More typically, theenriched nucleic acids represent 50% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules. In aone aspect, the enriched nucleic acids represent 90% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; i.e., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis. Solid-phase chemical peptide synthesismethods can also be used to synthesize the polypeptide or fragments ofthe invention. Such method have been known in the art since the early1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (Seealso Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recentlybeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate.

A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, refers to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one of the known sequencecomparison algorithms or by visual inspection. In alternative aspects,the substantial identity exists over a region of at least about 100 ormore residues and most commonly the sequences are substantiallyidentical over at least about 150 to 200 or more residues. In someaspects, the sequences are substantially identical over the entirelength of the coding regions.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule and provided that the polypeptideessentially retains its functional properties. A conservative amino acidsubstitution, for example, substitutes one amino acid for another of thesame class (e.g., substitution of one hydrophobic amino acid, such asisoleucine, valine, leucine, or methionine, for another, or substitutionof one polar amino acid for another, such as substitution of argininefor lysine, glutamic acid for aspartic acid or glutamine forasparagine). One or more amino acids can be deleted, for example, from alaccase polypeptide, resulting in modification of the structure of thepolypeptide, without significantly altering its biological activity. Forexample, amino- or carboxyl-terminal amino acids that are not requiredfor laccase biological activity can be removed. Modified polypeptidesequences of the invention can be assayed for laccase biologicalactivity by any number of methods, including contacting the modifiedpolypeptide sequence with a laccase substrate and determining whetherthe modified polypeptide decreases the amount of specific substrate inthe assay or increases the bioproducts of the enzymatic reaction of afunctional laccase polypeptide with the substrate.

“Fragments” as used herein are a portion of a naturally occurringprotein which can exist in at least two different conformations.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. Fragments which havedifferent three dimensional structures as the naturally occurringprotein are also included. An example of this, is a “pro-form” molecule,such as a low activity proprotein that can be modified by cleavage toproduce a mature enzyme with significantly higher activity.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. In alternativeaspects, nucleic acids of the invention are defined by their ability tohybridize under various stringency conditions (e.g., high, medium, andlow), as set forth herein.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In one aspect, hybridization occurs under highstringency conditions, e.g., at 42° C. in 50% formamide, 5×SSPE, 0.3%SDS and 200 n/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under these reduced stringency conditions, but in 35%formamide at a reduced temperature of 35° C. The temperature rangecorresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “variant” refers to polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of a laccase of the invention. Variants can be produced by anynumber of means included methods such as, for example, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, GSSM™ and any combination thereof.

The term “saturation mutagenesis”, Gene Site Saturation Mutagenesis™, or“GSSM™” includes a method that uses degenerate oligonucleotide primersto introduce point mutations into a polynucleotide, as described indetail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Nucleic Acids

The invention provides nucleic acids (e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQID NO:25; nucleic acids encoding polypeptides as set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24 or SEQ ID NO:26) including expression cassettes such asexpression vectors, encoding the polypeptides of the invention. Theinvention also includes methods for discovering new laccase sequencesusing the nucleic acids of the invention. The invention also includesmethods for inhibiting the expression of laccase genes, transcripts andpolypeptides using the nucleic acids of the invention. Also provided aremethods for modifying the nucleic acids of the invention by, e.g.,synthetic ligation reassembly, optimized directed evolution systemand/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Forexample, exemplary sequences of the invention were initially derivedfrom sources as set forth in Table 1, above.

In one aspect, the invention provides laccase-encoding nucleic acids,and the polypeptides encoded by them, with a common novelty in that theyare derived from a common source, e.g., an environmental or a bacterialsource.

In practicing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

One aspect of the invention is an isolated nucleic acid comprising oneof the sequences of the invention, or a fragment comprising at least 10,15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or moreconsecutive bases of a nucleic acid of the invention. The isolated,nucleic acids may comprise DNA, including cDNA, genomic DNA andsynthetic DNA. The DNA may be double-stranded or single-stranded and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. Alternatively, the isolated nucleic acids may comprise RNA.

The isolated nucleic acids of the invention may be used to prepare oneof the polypeptides of the invention, or fragments comprising at least5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutiveamino acids of one of the polypeptides of the invention. Accordingly,another aspect of the invention is an isolated nucleic acid whichencodes one of the polypeptides of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150or more consecutive amino acids of one of the polypeptides of theinvention. The coding sequences of these nucleic acids may be identicalto one of the coding sequences of one of the nucleic acids of theinvention or may be different coding sequences which encode one of theof the invention having at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 or more consecutive amino acids of one of the polypeptidesof the invention, as a result of the redundancy or degeneracy of thegenetic code. The genetic code is well known to those of skill in theart and can be obtained, e.g., on page 214 of B. Lewin, Genes VI, OxfordUniversity Press, 1997.

The isolated nucleic acid which encodes one of the polypeptides of theinvention, but is not limited to: only the coding sequence of a nucleicacid of the invention and additional coding sequences, such as leadersequences or proprotein sequences and non-coding sequences, such asintrons or non-coding sequences 5′ and/or 3′ of the coding sequence.Thus, as used herein, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only the coding sequence forthe polypeptide as well as a polynucleotide which includes additionalcoding and/or non-coding sequence.

Alternatively, the nucleic acid sequences of the invention, may bemutagenized using conventional techniques, such as site directedmutagenesis, or other techniques familiar to those skilled in the art,to introduce silent changes into the polynucleotides o of the invention.As used herein, “silent changes” include, for example, changes which donot alter the amino acid sequence encoded by the polynucleotide. Suchchanges may be desirable in order to increase the level of thepolypeptide produced by host cells containing a vector encoding thepolypeptide by introducing codons or codon pairs which occur frequentlyin the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of the invention. Suchnucleotide changes may be introduced using techniques such as sitedirected mutagenesis, random chemical mutagenesis, exonuclease IIIdeletion and other recombinant DNA techniques. Alternatively, suchnucleotide changes may be naturally occurring allelic variants which areisolated by identifying nucleic acids which specifically hybridize toprobes comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 300, 400, or 500 consecutive bases of one of the sequences of theinvention (or the sequences complementary thereto) under conditions ofhigh, moderate, or low stringency as provided herein.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., laccases) generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system can be used, includingbacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or tip promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused. Promoters suitable for expressing the polypeptide or fragmentthereof in bacteria include the E. coli lac or trp promoters, the ladpromoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gptpromoter, the lambda P_(R) promoter, the lambda P_(L) promoter,promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter.Fungal promoters include the α-factor promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express a laccase of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express a laccase of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Riot 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression oflaccase-expressing nucleic acid in a specific tissue, organ or cell type(i.e. tissue-specific promoters) or may be otherwise under more preciseenvironmental or developmental control or under the control of aninducible promoter. Examples of environmental conditions that may affecttranscription include anaerobic conditions, elevated temperature, thepresence of light, or sprayed with chemicals/hormones. For example, theinvention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the laccase-producing nucleic acids of the invention willallow the grower to select plants with the optimal laccase expressionand/or activity. The development of plant parts can thus controlled. Inthis way the invention provides the means to facilitate the harvestingof plants and plant parts. For example, in various embodiments, themaize In2-2 promoter, activated by benzenesulfonamide herbicidesafeners, is used (De Veylder (1997) Plant Cell Physiol. 38:568-577);application of different herbicide safeners induces distinct geneexpression patterns, including expression in the root, hydathodes, andthe shoot apical meristem. Coding sequences of the invention are alsounder the control of a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324).

In some aspects, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thelaccases of the invention. Expression vectors and cloning vehicles ofthe invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA that can be from about 10 toabout 300 bp in length. They can act on a promoter to increase itstranscription. Exemplary enhancers include the SV40 enhancer on the lateside of the replication origin bp 100 to 270, the cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and the adenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(I), and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors in one aspect contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences and 5′flanking nontranscribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin and theadenovirus enhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli and the S. cerevisiae TRP1 gene.

In some aspects, the nucleic acid encoding one of the polypeptides ofthe invention, or fragments comprising at least about 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.Optionally, the nucleic acid can encode a fusion polypeptide in whichone of the polypeptides of the invention, or fragments comprising atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof is fused to heterologous peptides or polypeptides,such as N-terminal identification peptides which impart desiredcharacteristics, such as increased stability or simplified purification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a laccase of theinvention, or a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Streptomyces, Bacillus subtilis,Bacillus cereus, Salmonella typhimurium and various species within thegenera Streptomyces and Staphylococcus. Exemplary insect cells includeDrosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO,COS or Bowes melanoma or any mouse or human cell line. The selection ofan appropriate host is within the abilities of those skilled in the art.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477; U.S. Pat. No.5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets can be used.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Host cells containing the polynucleotides of interest, e.g., nucleicacids of the invention, can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan. The clones which are identified as having the specifiedenzyme activity may then be sequenced to identify the polynucleotidesequence encoding an enzyme having the enhanced activity.

The invention provides a method for overexpressing a recombinant laccasein a cell comprising expressing a vector comprising a nucleic acid ofthe invention, e.g., a nucleic acid comprising a nucleic acid sequencewith at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to an exemplary sequence of the invention over aregion of at least about 100 residues, wherein the sequence identitiesare determined by analysis with a sequence comparison algorithm or byvisual inspection, or, a nucleic acid that hybridizes under stringentconditions to a nucleic acid sequence of the invention. Theoverexpression can be effected by any means, e.g., use of a highactivity promoter, a dicistronic vector or by gene amplification of thevector.

The nucleic acids of the invention can be expressed, or overexpressed,in any in vitro or in vivo expression system. Any cell culture systemscan be employed to express, or over-express, recombinant protein,including bacterial, insect, yeast, fungal or mammalian cultures.Over-expression can be effected by appropriate choice of promoters,enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors(see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun. 229:295-8),media, culture systems and the like. In one aspect, gene amplificationusing selection markers, e.g., glutamine synthetase (see, e.g., Sanders(1987) Dev. Biol. Stand. 66:55-63), in cell systems are used tooverexpress the polypeptides of the invention.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Streptomyces and Staphylococcus,fungal cells, such as yeast, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981) and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150or more consecutive amino acids thereof can be synthetically produced byconventional peptide synthesizers. In other aspects, fragments orportions of the polypeptides may be employed for producing thecorresponding full-length polypeptide by peptide synthesis; therefore,the fragments may be employed as intermediates for producing thefull-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of the invention, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive aminoacids thereof using mRNAs transcribed from a DNA construct comprising apromoter operably linked to a nucleic acid encoding the polypeptide orfragment thereof. In some aspects, the DNA construct may be linearizedprior to conducting an in vitro transcription reaction. The transcribedmRNA is then incubated with an appropriate cell-free translationextract, such as a rabbit reticulocyte extract, to produce the desiredpolypeptide or fragment thereof.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the laccases of the invention, or modified nucleic acidsof the invention, can be reproduced by amplification. Amplification canalso be used to clone or modify the nucleic acids of the invention.Thus, the invention provides amplification primer sequence pairs foramplifying nucleic acids of the invention. One of skill in the art candesign amplification primer sequence pairs for any part of or the fulllength of these sequences.

In one aspect, the invention provides a nucleic acid amplified by aprimer pair of the invention, e.g., a primer pair as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 or more residues of a nucleic acid of the invention, and about thefirst (the 5′) 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or moreresidues of the complementary strand.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a laccaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 or more consecutive bases of the sequence, or about 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more consecutive basesof the sequence. The invention provides amplification primer pairs,wherein the primer pair comprises a first member having a sequence asset forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 or more residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 or more residues of the complementary strand of the first member.The invention provides laccases generated by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. The invention provides methods of making a laccase byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity (homology) to an exemplary nucleic acid of theinvention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25) over a region of atleast about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues. Theinvention provides polypeptides comprising sequences having at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to an exemplary polypeptide of the invention (e.g.,SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24 or SEQ ID NO:26). The extent of sequence identity(homology) may be determined using any computer program and associatedparameters, including those described herein, such as BLAST 2.2.2. orFASTA version 3.0t78, with the default parameters.

Nucleic acid sequences of the invention can comprise at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or moreconsecutive nucleotides of an exemplary sequence of the invention andsequences substantially identical thereto. Homologous sequences andfragments of nucleic acid sequences of the invention can refer to asequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity (homology) to these sequences. Homology (sequenceidentity) may be determined using any of the computer programs andparameters described herein, including FASTA version 3.0t78 with thedefault parameters. Homologous sequences also include RNA sequences inwhich uridines replace the thymines in the nucleic acid sequences of theinvention. The homologous sequences may be obtained using any of theprocedures described herein or may result from the correction of asequencing error. It will be appreciated that the nucleic acid sequencesof the invention can be represented in the traditional single characterformat (See the inside back cover of Stryer, Lubert. Biochemistry, 3rdEd., W. H Freeman & Co., New York.) or in any other format which recordsthe identity of the nucleotides in a sequence.

Various sequence comparison programs identified elsewhere in this patentspecification are particularly contemplated for use in this aspect ofthe invention. Protein and/or nucleic acid sequence homologies may beevaluated using any of the variety of sequence comparison algorithms andprograms known in the art. Such algorithms and programs include, but areby no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW(see, e.g., Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al.,Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol.215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol 48:443, 1970, bythe search for similarity method of person & Lipman, Proc. Nat'l. Acad.Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). At least twenty-one other genomes have already beensequenced, including, for example, M. genitalium (Fraser et al., 1995),M. jannaschii (Bult et al., 1996), H. influenzae (Fleischmann et al.,1995), E. coli (Blattner et al., 1997) and yeast (S. cerevisiae) (Meweset al., 1997) and D. melanogaster (Adams et al., 2000). Significantprogress has also been made in sequencing the genomes of model organism,such as mouse, C. elegans and Arabadopsis sp. Several databasescontaining genomic information annotated with some functionalinformation are maintained by different organizations and may beaccessible via the internet.

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402,1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more in one aspect lessthan about 0.01 and most in one aspect less than about 0.001.

In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis in one aspect obtained from a protein or nucleic acid sequencedatabase. High-scoring segment pairs are in one aspect identified (i.e.,aligned) by means of a scoring matrix, many of which are known in theart. In one aspect, the scoring matrix used is the BLOSUM62 matrix(Gonnet (1992) Science 256:1443-1445; Henikoff and Henikoff (1993)Proteins 17:49-61). Less in one aspect, the PAM or PAM250 matrices mayalso be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, a nucleic acid or polypeptide sequence ofthe invention can be stored, recorded, and manipulated on any mediumwhich can be read and accessed by a computer.

Accordingly, the invention provides computers, computer systems,computer readable mediums, computer programs products and the likerecorded or stored thereon the nucleic acid and polypeptide sequences ofthe invention. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any known methods for recording information ona computer readable medium to generate manufactures comprising one ormore of the nucleic acid and/or polypeptide sequences of the invention.

The polypeptides of the invention include the polypeptide sequences ofthe invention, e.g., the exemplary sequences of the invention, andsequences substantially identical thereto, and fragments of any of thepreceding sequences. Substantially identical, or homologous, polypeptidesequences refer to a polypeptide sequence having at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity(homology) to an exemplary sequence of the invention.

Homology (sequence identity) may be determined using any of the computerprograms and parameters described herein. A nucleic acid or polypeptidesequence of the invention can be stored, recorded and manipulated on anymedium which can be read and accessed by a computer. As used herein, thewords “recorded” and “stored” refer to a process for storing informationon a computer medium. A skilled artisan can readily adopt any of thepresently known methods for recording information on a computer readablemedium to generate manufactures comprising one or more of the nucleicacid sequences of the invention, one or more of the polypeptidesequences of the invention. Another aspect of the invention is acomputer readable medium having recorded thereon at least 2, 5, 10, 15,or 20 or more nucleic acid or polypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences of theinvention. Another aspect of the invention is a computer readable mediumhaving recorded thereon one or more of the polypeptide sequences of theinvention. Another aspect of the invention is a computer readable mediumhaving recorded thereon at least 2, 5, 10, 15, or 20 or more of thenucleic acid or polypeptide sequences as set forth above.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems which store and manipulate the sequenceinformation described herein. One example of a computer system 100 isillustrated in block diagram form in FIG. 1. As used herein, “a computersystem” refers to the hardware components, software components and datastorage components used to analyze a nucleotide sequence of a nucleicacid sequence of the invention, or a polypeptide sequence of theinvention. The computer system 100 typically includes a processor forprocessing, accessing and manipulating the sequence data. The processor105 can be any well-known type of central processing unit, such as, forexample, the Pentium III from Intel Corporation, or similar processorfrom Sun, Motorola, Compaq, AMD or International Business Machines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular aspect, the computer system 100 includes a processor105 connected to a bus which is connected to a main memory 115 (in oneaspect implemented as RAM) and one or more internal data storage devices110, such as a hard drive and/or other computer readable media havingdata recorded thereon. In some aspects, the computer system 100 furtherincludes one or more data retrieving device 118 for reading the datastored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some aspects, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence of the invention, or a polypeptide sequence of theinvention, (such as search tools, compare tools and modeling tools etc.)may reside in main memory 115 during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence of theinvention, or a polypeptide sequence of the invention, stored on acomputer readable medium to a reference nucleotide or polypeptidesequence(s) stored on a computer readable medium. A “sequence comparisonalgorithm” refers to one or more programs which are implemented (locallyor remotely) on the computer system 100 to compare a nucleotide sequencewith other nucleotide sequences and/or compounds stored within a datastorage means. For example, the sequence comparison algorithm maycompare the nucleotide sequences of a nucleic acid sequence of theinvention, or a polypeptide sequence of the invention, stored on acomputer readable medium to reference sequences stored on a computerreadable medium to identify homologies or structural motifs.

FIG. 2 is a flow diagram illustrating one aspect of a process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence of the invention, or a polypeptide sequence of the invention, adata storage device having retrievably stored thereon referencenucleotide sequences or polypeptide sequences to be compared to anucleic acid sequence of the invention, or a polypeptide sequence of theinvention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs in the above described nucleicacid code a nucleic acid sequence of the invention, or a polypeptidesequence of the invention, or it may identify structural motifs insequences which are compared to these nucleic acid codes and polypeptidecodes. In some aspects, the data storage device may have stored thereonthe sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of thenucleic acid sequences of the invention, or the polypeptide sequences ofthe invention.

Another aspect of the invention is a method for determining the level ofhomology between a nucleic acid sequence of the invention, or apolypeptide sequence of the invention and a reference nucleotidesequence. The method including reading the nucleic acid code or thepolypeptide code and the reference nucleotide or polypeptide sequencethrough the use of a computer program which determines homology levelsand determining homology between the nucleic acid code or polypeptidecode and the reference nucleotide or polypeptide sequence with thecomputer program. The computer program may be any of a number ofcomputer programs for determining homology levels, including thosespecifically enumerated herein, (e.g., BLAST2N with the defaultparameters or with any modified parameters). The method may beimplemented using the computer systems described above. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 ormore of the above described nucleic acid sequences of the invention, orthe polypeptide sequences of the invention through use of the computerprogram and determining homology between the nucleic acid codes orpolypeptide codes and reference nucleotide sequences or polypeptidesequences.

FIG. 3 is a flow diagram illustrating one aspect of a process 250 in acomputer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is in one aspect in the single letter aminoacid code so that the first and sequence sequences can be easilycompared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code of the invention,differs from a reference nucleic acid sequence at one or more positions.Optionally such a program records the length and identity of inserted,deleted or substituted nucleotides with respect to the sequence ofeither the reference polynucleotide or a nucleic acid sequence of theinvention. In one aspect, the computer program may be a program whichdetermines whether a nucleic acid sequence of the invention, contains asingle nucleotide polymorphism (SNP) with respect to a referencenucleotide sequence.

Accordingly, another aspect of the invention is a method for determiningwhether a nucleic acid sequence of the invention, differs at one or morenucleotides from a reference nucleotide sequence comprising the steps ofreading the nucleic acid code and the reference nucleotide sequencethrough use of a computer program which identifies differences betweennucleic acid sequences and identifying differences between the nucleicacid code and the reference nucleotide sequence with the computerprogram. In some aspects, the computer program is a program whichidentifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acidsequences of the invention and the reference nucleotide sequencesthrough the use of the computer program and identifying differencesbetween the nucleic acid codes and the reference nucleotide sequenceswith the computer program.

In other aspects the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence ofthe invention or a polypeptide sequence of the invention.

An “identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence of the invention, or apolypeptide sequence of the invention. In one aspect, the identifier maycomprise a program which identifies an open reading frame in a nucleicacid sequence of the invention.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic active sites,helix-turn-helix motifs or other motifs known to those skilled in theart.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence. It should be noted, that if thefeature attribute is not found in the first sequence at the decisionstate 316, the process 300 moves directly to the decision state 320 inorder to determine if any more features exist in the database.

Accordingly, another aspect of the invention is a method of identifyinga feature within a nucleic acid sequence of the invention, or apolypeptide sequence of the invention, comprising reading the nucleicacid code(s) or polypeptide code(s) through the use of a computerprogram which identifies features therein and identifying featureswithin the nucleic acid code(s) with the computer program. In oneaspect, computer program comprises a computer program which identifiesopen reading frames. The method may be performed by reading a singlesequence or at least 2, 5, 10, 15, 20, 25, 30, or 40 of the nucleic acidsequences of the invention, or the polypeptide sequences of theinvention, through the use of the computer program and identifyingfeatures within the nucleic acid codes or polypeptide codes with thecomputer program.

A nucleic acid sequence of the invention, or a polypeptide sequence ofthe invention, may be stored and manipulated in a variety of dataprocessor programs in a variety of formats. For example, a nucleic acidsequence of the invention, or a polypeptide sequence of the invention,may be stored as text in a word processing file, such as Microsoft WORD™or WORDPERFECT™ or as an ASCII file in a variety of database programsfamiliar to those of skill in the art, such as DB2™, SYBASE™, orORACLE™. In addition, many computer programs and databases may be usedas sequence comparison algorithms, identifiers, or sources of referencenucleotide sequences or polypeptide sequences to be compared to anucleic acid sequence of the invention, or a polypeptide sequence of theinvention. The following list is intended not to limit the invention butto provide guidance to programs and databases which are useful with thenucleic acid sequences of the invention, or the polypeptide sequences ofthe invention.

The programs and databases which may be used include, but are notlimited to: MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARMm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwents'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25). The stringentconditions can be highly stringent conditions, medium stringentconditions and/or low stringent conditions, including the high andreduced stringency conditions described herein. In one aspect, it is thestringency of the wash conditions that set forth the conditions whichdetermine whether a nucleic acid is within the scope of the invention,as discussed below.

In alternative aspects, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA (single or double stranded),antisense or sequences encoding antibody binding peptides (epitopes),motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent) and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

All of the foregoing hybridizations would be considered to be underconditions of high stringency.

Following hybridization, a filter can be washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content) and the nucleic acid type (e.g., RNA v. DNA). Examples ofprogressively higher stringency condition washes are as follows: 2×SSC,0.1% SDS at room temperature for 15 minutes (low stringency); 0.1×SSC,0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

In one aspect, hybridization conditions comprise a wash step comprisinga wash for 30 minutes at room temperature in a solution comprising 1×150mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na₂EDTA, 0.5% SDS,followed by a 30 minute wash in fresh solution.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention. Forexample, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 97%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% sequence identity (homology) toa nucleic acid sequence selected from the group consisting of one of thesequences of the invention, or fragments comprising at least about 10,15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500consecutive bases thereof and the sequences complementary thereto.Sequence identity (homology) may be measured using the alignmentalgorithm. For example, the homologous polynucleotides may have a codingsequence which is a naturally occurring allelic variant of one of thecoding sequences described herein. Such allelic variants may have asubstitution, deletion or addition of one or more nucleotides whencompared to the nucleic acids of the invention. Additionally, the aboveprocedures may be used to isolate nucleic acids which encodepolypeptides having at least about 99%, 95%, at least 90%, at least 85%,at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, or at least 50% sequence identity (homology) to a polypeptideof the invention, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof asdetermined using a sequence alignment algorithm (e.g., such as the FASTAversion 3.0t78 algorithm with the default parameters).

Oligonucleotides Probes and Methods for Using Them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with a laccaseactivity or fragments thereof or for identifying laccase genes. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutivebases of a sequence as set forth in a nucleic acid of the invention. Theprobes identify a nucleic acid by binding and/or hybridization. Theprobes can be used in arrays of the invention, see discussion below,including, e.g., capillary arrays. The probes of the invention can alsobe used to isolate other nucleic acids or polypeptides.

The isolated nucleic acids of the invention, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of the invention, or the sequences complementary thereto mayalso be used as probes to determine whether a biological sample, such asa soil sample, contains an organism having a nucleic acid sequence ofthe invention or an organism from which the nucleic acid was obtained.In such procedures, a biological sample potentially harboring theorganism from which the nucleic acid was isolated is obtained andnucleic acids are obtained from the sample. The nucleic acids arecontacted with the probe under conditions which permit the probe tospecifically hybridize to any complementary sequences from which arepresent therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress (1989.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook,supra. Alternatively, the amplification may comprise a ligase chainreaction, 3SR, or strand displacement reaction. (See Barany, F., “TheLigase Chain Reaction in a PCR World”, PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication(3SR): An Isothermal Transcription-based Amplification SystemAlternative to PCR”, PCR Methods and Applications 1:25-33, 1991; andWalker G. T. et al., “Strand Displacement Amplification—an Isothermal invitro DNA Amplification Technique”, Nucleic Acid Research 20:1691-1696,1992). In such procedures, the nucleic acids in the sample are contactedwith the probes, the amplification reaction is performed and anyresulting amplification product is detected. The amplification productmay be detected by performing gel electrophoresis on the reactionproducts and staining the gel with an intercalator such as ethidiumbromide. Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences of theinvention, may also be used in chromosome walking procedures to identifyclones containing genomic sequences located adjacent to the sequences ofthe invention. Such methods allow the isolation of genes which encodeadditional proteins from the host organism.

The isolated nucleic acids of the invention, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of the invention, or the sequences complementary thereto maybe used as probes to identify and isolate related nucleic acids. In someaspects, the related nucleic acids may be cDNAs or genomic DNAs fromorganisms other than the one from which the nucleic acid was isolated.For example, the other organisms may be related organisms. In suchprocedures, a nucleic acid sample is contacted with the probe underconditions which permit the probe to specifically hybridize to relatedsequences. Hybridization of the probe to nucleic acids from the relatedorganism is then detected using any of the methods described above.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50%formamide. The formulas for SSC and Denhardt's solutions are listed inSambrook et al., supra.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T_(m). In one aspect, for hybridizationsin 6×SSC, the hybridization is conducted at approximately 68° C.Usually, for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

Inhibiting Expression of Laccases

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g., laccase-encodingnucleic acids, e.g., nucleic acids comprising antisense, iRNA,ribozymes. Nucleic acids of the invention comprising antisense sequencescan be capable of inhibiting the transport, splicing or transcription oflaccase-encoding genes. The inhibition can be effected through thetargeting of genomic DNA or messenger RNA. The transcription or functionof targeted nucleic acid can be inhibited, for example, by hybridizationand/or cleavage. One particularly useful set of inhibitors provided bythe present invention includes oligonucleotides which are able to eitherbind laccase gene or message, in either case preventing or inhibitingthe production or function of laccase. The association can be throughsequence specific hybridization. Another useful class of inhibitorsincludes oligonucleotides which cause inactivation or cleavage oflaccase message. The oligonucleotide can have enzyme activity whichcauses such cleavage, such as ribozymes. The oligonucleotide can bechemically modified or conjugated to an enzyme or composition capable ofcleaving the complementary nucleic acid. A pool of many different sucholigonucleotides can be screened for those with the desired activity.Thus, the invention provides various compositions for the inhibition oflaccase expression on a nucleic acid and/or protein level, e.g.,antisense, iRNA and ribozymes comprising laccase sequences of theinvention and the anti-laccase antibodies of the invention.

Inhibition of laccase expression can have a variety of industrialapplications. For example, inhibition of laccase expression can slow orprevent spoilage. In one aspect, use of compositions of the inventionthat inhibit the expression and/or activity of laccases, e.g.,antibodies, antisense oligonucleotides, ribozymes and RNAi, are used toslow or prevent spoilage. Thus, in one aspect, the invention providesmethods and compositions comprising application onto a plant or plantproduct (e.g., a cereal, a grain, a fruit, seed, root, leaf, etc.)antibodies, antisense oligonucleotides, ribozymes and RNAi of theinvention to slow or prevent spoilage. These compositions also can beexpressed by the plant (e.g., a transgenic plant) or another organism(e.g., a bacterium or other microorganism transformed with a laccasegene of the invention).

The compositions of the invention for the inhibition of laccaseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions, e.g., as anti-pathogen agents or in othertherapies, e.g., as anti-microbials for, e.g., Salmonella.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindinglaccase message which, in one aspect, can inhibit laccase activity bytargeting mRNA. Strategies for designing antisense oligonucleotides arewell described in the scientific and patent literature, and the skilledartisan can design such laccase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisense laccasesequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem.270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding laccase message.These ribozymes can inhibit laccase activity by, e.g., targeting mRNA.Strategies for designing ribozymes and selecting the laccase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it canbe released from that RNA to bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a laccase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of a laccase gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a laccase. These methodscan be repeated or used in various combinations to generate laccaseshaving an altered or different activity or an altered or differentstability from that of a laccase encoded by the template nucleic acid.These methods also can be repeated or used in various combinations,e.g., to generate variations in gene/message expression, messagetranslation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis™ (GSSM™), synthetic ligation reassembly(SLR), recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation, and/or a combination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis, such as Gene Site Saturation Mutagenesis™(GSSM™), synthetic ligation reassembly (SLR), or a combination thereofare used to modify the nucleic acids of the invention to generatelaccases with new or altered properties (e.g., activity under highlyacidic or alkaline conditions, high or low temperatures, and the like).Polypeptides encoded by the modified nucleic acids can be screened foran activity before testing for glucan hydrolysis or other activity. Anytesting modality or protocol can be used, e.g., using a capillary arrayplatform. See, e.g., U.S. Pat. Nos. 6,361,974; 6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM™

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., alaccase or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,laccases) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other aspects useless than all 20 natural combinations). The 32-fold degenerate progenypolypeptides generated from each saturation mutagenesis reaction vesselcan be subjected to clonal amplification (e.g. cloned into a suitablehost, e.g., E. coli host, using, e.g., an expression vector) andsubjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedglucan hydrolysis activity under alkaline or acidic conditions), it canbe sequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (Gene Site Saturation Mutagenesis™ (GSSM™)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and in one aspect but notnecessarily a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprised of only one N, where the N canbe in the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N,G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N,G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N,G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changesand point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in one aspect of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is in one aspect everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(in one aspect a subset totaling from 15 to 100,000) to mutagenesis. Inone aspect, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations can beintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette is in oneaspect about 1-500 bases in length) in defined polynucleotide sequenceto be mutagenized (wherein the sequence to be mutagenized is in oneaspect from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons and groupings ofparticular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF) and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one exemplification a grouping of mutations that can be introducedinto a mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., laccases or antibodies of theinvention, with new or altered properties.

SLR is a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. patent application Ser. No.09/332,835 entitled “Synthetic Ligation Reassembly in DirectedEvolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). Inone aspect, SLR comprises the following steps: (a) providing a templatepolynucleotide, wherein the template polynucleotide comprises sequenceencoding a homologous gene; (b) providing a plurality of building blockpolynucleotides, wherein the building block polynucleotides are designedto cross-over reassemble with the template polynucleotide at apredetermined sequence, and a building block polynucleotide comprises asequence that is a variant of the homologous gene and a sequencehomologous to the template polynucleotide flanking the variant sequence;(c) combining a building block polynucleotide with a templatepolynucleotide such that the building block polynucleotide cross-overreassembles with the template polynucleotide to generate polynucleotidescomprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are in one aspect shared by atleast two of the progenitor templates. The demarcation points canthereby be used to delineate the boundaries of oligonucleotide buildingblocks to be generated in order to rearrange the parentalpolynucleotides. The demarcation points identified and selected in theprogenitor molecules serve as potential chimerization points in theassembly of the final chimeric progeny molecules. A demarcation pointcan be an area of homology (comprised of at least one homologousnucleotide base) shared by at least two parental polynucleotidesequences. Alternatively, a demarcation point can be an area of homologythat is shared by at least half of the parental polynucleotidesequences, or, it can be an area of homology that is shared by at leasttwo thirds of the parental polynucleotide sequences. Even more in oneaspect a serviceable demarcation points is an area of homology that isshared by at least three fourths of the parental polynucleotidesequences, or, it can be shared by at almost all of the parentalpolynucleotide sequences. In one aspect, a demarcation point is an areaof homology that is shared by all of the parental polynucleotidesequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedin one aspect comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In one aspect, the present invention provides a non-stochastic methodtermed synthetic gene reassembly, that is somewhat related to stochasticshuffling, save that the nucleic acid building blocks are not shuffledor concatenated or chimerized randomly, but rather are assemblednon-stochastically.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one aspect of the invention, the annealedbuilding pieces are treated with an enzyme, such as a ligase (e.g., T4DNA ligase) to achieve covalent bonding of the building pieces.

In a another aspect, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Thelaccases of the present invention can be mutagenized in accordance withthe methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., polynucleotides ofthe invention) are aligned in order to select one or more demarcationpoints, which demarcation points can be located at an area of homology.The demarcation points can be used to delineate the boundaries ofnucleic acid building blocks to be generated. Thus, the demarcationpoints identified and selected in the progenitor molecules serve aspotential chimerization points in the assembly of the progeny molecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates and in one aspect at almost all of theprogenitor templates. Even more in one aspect still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

In a one aspect, the gene reassembly process is performed exhaustivelyin order to generate an exhaustive library. In other words, all possibleordered combinations of the nucleic acid building blocks are representedin the set of finalized chimeric nucleic acid molecules. At the sametime, the assembly order (i.e. the order of assembly of each buildingblock in the 5′ to 3 sequence of each finalized chimeric nucleic acid)in each combination is by design (or non-stochastic). Because of thenon-stochastic nature of the method, the possibility of unwanted sideproducts is greatly reduced.

In another aspect, the method provides that the gene reassembly processis performed systematically, for example to generate a systematicallycompartmentalized library, with compartments that can be screenedsystematically, e.g., one by one. In other words the invention providesthat, through the selective and judicious use of specific nucleic acidbuilding blocks, coupled with the selective and judicious use ofsequentially stepped assembly reactions, an experimental design can beachieved where specific sets of progeny products are made in each ofseveral reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated in one aspect comprise a library offinalized chimeric nucleic acid molecules having an overall assemblyorder that is chosen by design. In a particularly aspect, such agenerated library is comprised of greater than 10³ to greater than10¹⁰⁰⁰ different progeny molecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one aspect, this polynucleotide is a gene,which may be a man-made gene. According to another aspect, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g., by mutagenesis) or in an in vivoprocess (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another aspect, the invention provides that a nucleicacid building block can be used to introduce an intron. Thus, theinvention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). In one aspect, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In one aspect,the recombination is facilitated by, or occurs at, areas of homologybetween the man-made, intron-containing gene and a nucleic acid, whichserves as a recombination partner. In one aspect, the recombinationpartner may also be a nucleic acid generated by the invention, includinga man-made gene or a man-made gene pathway. Recombination may befacilitated by or may occur at areas of homology that exist at the one(or more) artificially introduced intron(s) in the man-made gene.

The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which in one aspecthas two ligatable ends. The two ligatable ends on each nucleic acidbuilding block may be two blunt ends (i.e. each having an overhang ofzero nucleotides), or in one aspect one blunt end and one overhang, ormore in one aspect still two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

In one aspect, a nucleic acid building block is generated by chemicalsynthesis of two single-stranded nucleic acids (also referred to assingle-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Exemplarysizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherexemplary size ranges are also provided, which have lower limits of from1 bp to 10,000 bp (including every integer value in between) and upperlimits of from 2 bp to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one aspect, a double-stranded nucleic acid building blockis generated by first generating two single stranded nucleic acids andallowing them to anneal to form a double-stranded nucleic acid buildingblock. The two strands of a double-stranded nucleic acid building blockmay be complementary at every nucleotide apart from any that form anoverhang; thus containing no mismatches, apart from any overhang(s).According to another aspect, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisaspect, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. In one aspect the codon degeneracy isintroduced using the site-saturation mutagenesis described herein, usingone or more N,N,G/T cassettes or alternatively using one or more N,N,Ncassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect the invention described herein is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionwhich allow for the directed molecular evolution of highly complexlinear sequences, such as DNA, RNA or proteins thorough recombination.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,laccases or antibodies of the invention, with new or altered properties.Optimized directed evolution is directed to the use of repeated cyclesof reductive reassortment, recombination and selection that allow forthe directed molecular evolution of nucleic acids through recombination.Optimized directed evolution allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide in one aspect includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a 1/3 chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide in one aspectincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is in one aspect performed in MATLAB™ (The Mathworks, Natick,Mass.) a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new laccase phenotype is identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, e.g., laccase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new laccase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,laccases, and the like. In vivo shuffling can be performed utilizing thenatural property of cells to recombine multimers. While recombination invivo has provided the major natural route to molecular diversity,genetic recombination remains a relatively complex process thatinvolves 1) the recognition of homologies; 2) strand cleavage, strandinvasion, and metabolic steps leading to the production of recombinantchiasma; and finally 3) the resolution of chiasma into discreterecombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In another aspect, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide (e.g., one, or both, being an exemplarylaccase-encoding sequence of the invention) which share at least oneregion of partial sequence homology into a suitable host cell. Theregions of partial sequence homology promote processes which result insequence reorganization producing a hybrid polynucleotide. The term“hybrid polynucleotide”, as used herein, is any nucleotide sequencewhich results from the method of the present invention and containssequence from at least two original polynucleotide sequences. Suchhybrid polynucleotides can result from intermolecular recombinationevents which promote sequence integration between DNA molecules. Inaddition, such hybrid polynucleotides can result from intramolecularreductive reassortment processes which utilize repeated sequences toalter a nucleotide sequence within a DNA molecule.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, novel polynucleotides canbe generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNaseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by:

-   1) The use of vectors only stably maintained when the construct is    reduced in complexity.-   2) The physical recovery of shortened vectors by physical    procedures. In this case, the cloning vector would be recovered    using standard plasmid isolation procedures and size fractionated on    either an agarose gel, or column with a low molecular weight cut off    utilizing standard procedures.-   3) The recovery of vectors containing interrupted genes which can be    selected when insert size decreases.-   4) The use of direct selection techniques with an expression vector    and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are described. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence. The quasi-repeatedsequences are separately or collectively amplified and ligated intorandom assemblies such that all possible permutations and combinationsare available in the population of ligated molecules. The number ofquasi-repeat units can be controlled by the assembly conditions. Theaverage number of quasi-repeated units in a construct is defined as therepetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector and transfected into an appropriate host cell. The cells are thenpropagated and “reductive reassortment” is effected. The rate of thereductive reassortment process may be stimulated by the introduction ofDNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution and the like)and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”) andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Exemplary means for slowing or halting PCRamplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA adductsor polynucleotides comprising the DNA adducts from the polynucleotidesor polynucleotides pool, which can be released or removed by a processincluding heating the solution comprising the polynucleotides prior tofurther processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., laccase) sequences of the invention.The invention also provides additional methods for isolating laccasesusing the nucleic acids and polypeptides of the invention. In oneaspect, the invention provides for variants of a laccase coding sequence(e.g., a gene, cDNA or message) of the invention, which can be alteredby any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung (1989) Technique 1:11-15) and Caldwell(1992) PCR Methods Applic. 2:28-33. Briefly, in such procedures, nucleicacids to be mutagenized are mixed with PCR primers, reaction buffer,MgCl₂, MnCl₂, Taq polymerase and an appropriate concentration of dNTPsfor achieving a high rate of point mutation along the entire length ofthe PCR product. For example, the reaction may be performed using 20fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, areaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01%gelatin, 7 mM MgCl2, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mMdGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed for 30cycles of 94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min.However, it will be appreciated that these parameters may be varied asappropriate. The mutagenized nucleic acids are cloned into anappropriate vector and the activities of the polypeptides encoded by themutagenized nucleic acids are evaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis” and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis.

The variants of the polypeptides of the invention may be variants inwhich one or more of the amino acid residues of the polypeptides of thesequences of the invention are substituted with a conserved ornon-conserved amino acid residue (in one aspect a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Alanine,Valine, Leucine and Isoleucine with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue such as Aspartic acid and Glutamic acid with anotheracidic residue; replacement of a residue bearing an amide group, such asAsparagine and Glutamine, with another residue bearing an amide group;exchange of a basic residue such as Lysine and Arginine with anotherbasic residue; and replacement of an aromatic residue such asPhenylalanine, Tyrosine with another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof a polypeptide of the invention includes a substituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide.

In some aspects, the fragments, derivatives and analogs retain the samebiological function or activity as the polypeptides of the invention. Inother aspects, the fragment, derivative, or analog includes aproprotein, such that the fragment, derivative, or analog can beactivated by cleavage of the proprotein portion to produce an activepolypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying laccase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding a laccase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding a laccase modified to increase itsexpression in a host cell, laccase so modified, and methods of makingthe modified laccases. The method comprises identifying a“non-preferred” or a “less preferred” codon in laccase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli; gram positive bacteria, such as Streptomyces sp., Lactobacillusgasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis,Bacillus cereus. Exemplary host cells also include eukaryotic organisms,e.g., various yeast, such as Saccharomyces sp., including Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris, andKluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, andmammalian cells and cell lines and insect cells and cell lines. Thus,the invention also includes nucleic acids and polypeptides optimized forexpression in these organisms and species.

For example, the codons of a nucleic acid encoding a laccase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the laccase was derived, a yeast, a fungi, a plant cell, an insectcell or a mammalian cell. Methods for optimizing codons are well knownin the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., a laccase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides methods of making and using these transgenic non-humananimals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studylaccase activity, or, as models to screen for agents that change thelaccase activity in vivo. The coding sequences for the polypeptides tobe expressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice. U.S. Pat.No. 6,187,992, describes making and using a transgenic mouse.

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing a laccase of the invention, or, a fusion proteincomprising a laccase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a laccase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides plant products, e.g., oils, seeds, leaves, extracts andthe like, comprising a nucleic acid and/or a polypeptide (e.g., alaccase) of the invention. The transgenic plant can be dicotyledonous (adicot) or monocotyledonous (a monocot). The invention also providesmethods of making and using these transgenic plants and seeds. Thetransgenic plant or plant cell expressing a polypeptide of the presentinvention may be constructed in accordance with any method known in theart. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's laccase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of laccase. The can changelaccase activity in a plant. Alternatively, a laccase of the inventioncan be used in production of a transgenic plant to produce a compoundnot naturally produced by that plant. This can lower production costs orcreate a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., alaccase) of the invention. The desired effects can be passed to futureplant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., a laccase orantibody) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity, or homology) to an exemplary sequence of the invention, e.g.,proteins having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ IDNO:26). The percent sequence identity can be over the full length of thepolypeptide, or, the identity can be over a region of at least about 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700 or more residues.

Polypeptides of the invention can also be shorter than the full lengthof exemplary polypeptides. In alternative aspects, the inventionprovides polypeptides (peptides, fragments) ranging in size betweenabout 5 and the full length of a polypeptide, e.g., an enzyme, such as alaccase; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g.,contiguous residues of an exemplary laccase of the invention. Peptidesof the invention (e.g., a subsequence of an exemplary polypeptide of theinvention) can be useful as, e.g., labeling probes, antigens,toleragens, motifs, laccase active sites (e.g., “catalytic domains”),signal sequences and/or prepro domains.

In one aspect, the polypeptide has a laccase activity. In one aspect,laccase activity of the polypeptides of the invention comprisescatalysis of oxidation of dioxygen (O₂) to two molecules of water withsimultaneously one-electron oxidation of an aromatic substrate, e.g., apolyphenol, a methoxy-substituted monophenol, an aromatic amine, or anyoxidizable aromatic compound. In one aspect, the laccase activity of theinvention comprises catalysis of oxidization of a polyphenol, amethoxy-substituted monophenol, an aromatic amine, or any oxidizablearomatic compound.

In one aspect, the laccase activity comprises catalyzing the oxidationof lignin. In one aspect, the laccase activity comprises thedepolymerization or polymerization of lignin. In one aspect, the laccaseactivity comprises catalyzing the oxidation of 1-hydroxybenzotriazole(HBT), N-benzoyl-N-phenyl hydroxylamine (BPHA), N-hydroxyphthalimide,3-hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB),2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), dimethoxyphenol ordihydroxyfumaric acid (DHF) or equivalent compounds.

In alternative aspects, polypeptides of the invention having laccaseactivity are members of a genus of polypeptides sharing specificstructural elements, e.g., amino acid residues, that correlate withlaccase activity, e.g., catalysis of oxidation of an aromatic substrate,such as a polyphenol, a methoxy-substituted monophenol, an aromaticamine, or any oxidizable aromatic (e.g., phenolic) compound. Theseshared structural elements can be used for the routine generation oflaccase variants. For example, in one aspect, laccases have keycatalytic site residues, such as the tripeptide “HCH”, see, e.g.,Piontek (2002) J. Biol. Chem. 277(40):37663-37669. In some aspects, alaccase can have additional sites, where the cysteine can besubstituted, for example, HWH, HSH, HLH. Alignment of exemplarysequences of laccases the invention are illustrated in FIG. 10 and FIG.11 (the sequence alignments were done with ClustalW, default parameters,see discussion, above). These alignments illustrate exemplary sharedstructural elements of laccase sequences of the invention, e.g., thelaccase families of the invention, as set forth in FIG. 10 (SEQ ID NO:4,SEQ ID NO:8) and FIG. 11 (SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO:18, SEQID NO:20, SEQ ID NO:26, SEQ ID NO:14, SEQ ID NO:6); please note the“consensus” sequence line in both Figures. Each exemplary laccasecomprises an HCH tripeptide (near the end of each sequence), plus threeother HXH or HXXHXH combinations. These shared structural elements oflaccases of the invention can be used as guidance for the routinegeneration of laccase variants within the scope of the genus of laccasesof the invention.

Additionally, the crystal structure of some laccases has been analyzed,e.g., see Piontek (2002), supra; Antorini (2002) Biochim. Biophys. Acta.1594(1):109-114, illustrating specific structural elements for theroutine generation of laccase variants.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants or members of a genus of polypeptides of theinvention (e.g., having about 50% or more sequence identity to anexemplary sequence of the invention), routine experimentation willdetermine whether a mimetic is within the scope of the invention, i.e.,that its structure and/or function is not substantially altered. Thus,in one aspect, a mimetic composition is within the scope of theinvention if it has a laccase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, N.Y.).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generated bysubstitution with, e.g., (in addition to lysine and arginine) the aminoacids ornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, inone aspect under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The polypeptides of the invention include laccases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude laccases inactive for other reasons, e.g., before “activation”by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike. The polypeptides of the invention include all active forms,including active subsequences, e.g., catalytic domains or active sites,of the laccase.

The invention includes immobilized laccases, anti-laccase antibodies andfragments thereof. The invention provides methods for inhibiting laccaseactivity, e.g., using dominant negative mutants or anti-laccaseantibodies of the invention. The invention includes heterocomplexes,e.g., fusion proteins, heterodimers, etc., comprising the laccases ofthe invention.

Polypeptides of the invention can have a laccase activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativelaccase preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, laccase variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety oflaccase variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify laccase modulators, e.g., activators or inhibitors of laccaseactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to laccase assays to determine their ability to inhibitsubstrate cleavage Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith laccases, inhibitors can be combined to increase the spectrum ofactivity.

The enzymes of the invention are also useful as research reagents todigest proteins or in protein sequencing. For example, the laccases maybe used to break polypeptides into smaller fragments for sequencingusing, e.g. an automated sequencer.

The invention also provides methods of discovering new laccases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, phagemid libraries are screened for expression-based discoveryof laccases. In another aspect, lambda phage libraries are screened forexpression-based discovery of laccases. Screening of the phage orphagemid libraries can allow the detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of phage or phagemidlibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

In one aspect, polypeptides or fragments of the invention may beobtained through biochemical enrichment or purification procedures. Thesequence of potentially homologous polypeptides or fragments may bedetermined by laccase assays (see, e.g., Example 1, below), gelelectrophoresis and/or microsequencing. The sequence of the prospectivepolypeptide or fragment of the invention can be compared to an exemplarypolypeptide of the invention, or a fragment, e.g., comprising at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or moreconsecutive amino acids thereof using any of the programs describedabove.

Another aspect of the invention is an assay for identifying fragments orvariants of the invention, which retain the enzymatic function of thepolypeptides of the invention. For example the fragments or variants ofsaid polypeptides, may be used to catalyze biochemical reactions (e.g.,production of a nootkatone from a valencene), which indicate that thefragment or variant retains the enzymatic activity of a polypeptide ofthe invention.

An exemplary assay for determining if fragments of variants retain theenzymatic activity of the polypeptides of the invention includes thesteps of: contacting the polypeptide fragment or variant with asubstrate molecule under conditions which allow the polypeptide fragmentor variant to function and detecting either a decrease in the level ofsubstrate or an increase in the level of the specific reaction productof the reaction between the polypeptide and substrate.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups and can react with many startingcompounds containing this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods.

In a particular aspect, the invention provides a method for modifyingsmall molecules, comprising contacting a polypeptide encoded by apolynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity isoptionally repeated. The biocatalytic reactions are conducted with agroup of biocatalysts that react with distinct structural moieties foundwithin the structure of a small molecule, each biocatalyst is specificfor one structural moiety or a group of related structural moieties; andeach biocatalyst reacts with many different small molecules whichcontain the distinct structural moiety.

Laccase Signal Sequences, Prepro and Catalytic Domains

The invention provides laccase signal sequences (e.g., signal peptides(SPs)), prepro domains and catalytic domains (CDs). The SPs, preprodomains and/or CDs of the invention can be isolated or recombinantpeptides or can be part of a fusion protein, e.g., as a heterologousdomain in a chimeric protein. The invention provides nucleic acidsencoding these catalytic domains (CDs), prepro domains and signalsequences (SPs, e.g., a peptide having a sequence comprising/consistingof amino terminal residues of a polypeptide of the invention).

The invention provides isolated or recombinant signal sequences (e.g.,signal peptides) consisting of or comprising a sequence as set forth inresidues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20,1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 27, 1 to 28, 1 to 28, 1 to 30,1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38,1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, or 1 to47, or more, of a polypeptide of the invention, e.g., SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26. In one aspect, the invention provides signalsequences comprising the first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino terminalresidues of a polypeptide of the invention. In one aspect, the inventionprovides signal sequences as set forth in Table 1, above.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated orrecombinant signal sequences, prepro sequences and catalytic domains(e.g., “active sites”) comprising sequences of the invention. Thepolypeptide comprising a signal sequence of the invention can be alaccase of the invention or another laccase or another enzyme or otherpolypeptide.

The laccase signal sequences (SPs) and/or prepro sequences of theinvention can be isolated peptides, or, sequences joined to anotherlaccase or a non-laccase polypeptide, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisinglaccase signal sequences of the invention. In one aspect, polypeptidescomprising laccase signal sequences SPs and/or prepro of the inventioncomprise sequences heterologous to a laccase of the invention (e.g., afusion protein comprising an SP and/or prepro of the invention andsequences from another laccase or a non-laccase protein). In one aspect,the invention provides laccases of the invention with heterologous SPsand/or prepro sequences, e.g., sequences with a yeast signal sequence. Alaccase of the invention can comprise a heterologous SP and/or prepro ina vector, e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs and/or prepro sequences of the invention areidentified following identification of novel laccase polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. The signal sequences can vary in length fromabout 10 to 65, or more, amino acid residues. Various methods ofrecognition of signal sequences are known to those of skill in the art.For example, in one aspect, novel laccase signal peptides are identifiedby a method referred to as SignalP. SignalP uses a combined neuralnetwork which recognizes both signal peptides and their cleavage sites.(Nielsen (1997) “Identification of prokaryotic and eukaryotic signalpeptides and prediction of their cleavage sites.” Protein Engineering10:1-6.

It should be understood that in some aspects laccases of the inventionmay not have SPs and/or prepro sequences, or “domains.” In one aspect,the invention provides the laccases of the invention lacking all or partof an SP and/or a prepro domain. In one aspect, the invention provides anucleic acid sequence encoding a signal sequence (SP) and/or prepro fromone laccase operably linked to a nucleic acid sequence of a differentlaccase or, optionally, a signal sequence (SPs) and/or prepro domainfrom a non-laccase protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa laccase) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated or recombinant polypeptide comprising (orconsisting of) a polypeptide comprising a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention with the provisothat it is not associated with any sequence to which it is naturallyassociated (e.g., a laccase sequence). Similarly in one aspect, theinvention provides isolated or recombinant nucleic acids encoding thesepolypeptides. Thus, in one aspect, the isolated or recombinant nucleicacid of the invention comprises coding sequence for a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)of the invention). The heterologous sequence can be on the 3′ terminalend, 5′ terminal end, and/or on both ends of the SP, prepro domainand/or CD coding sequence.

Hybrid (Chimeric) Laccases and Peptide Libraries

In one aspect, the invention provides hybrid laccases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such aslaccase substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like. In one aspect,the invention provides chimeric proteins comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention or acombination thereof and a heterologous sequence (see above).

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of laccases of the invention and other peptides, including knownand random peptides. They can be fused in such a manner that thestructure of the laccases is not significantly perturbed and the peptideis metabolically or structurally conformationally stabilized. Thisallows the creation of a peptide library that is easily monitored bothfor its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of a laccase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed laccase variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using, e.g., assays ofglucan hydrolysis. In alternative aspects, amino acid substitutions canbe single residues; insertions can be on the order of from about 1 to 20amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides laccases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e., a laccase activity) although variants can be selected tomodify the characteristics of the laccases as needed.

In one aspect, laccases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the laccases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the laccase are linkedtogether, in such a manner as to minimize the disruption to thestability of the laccase structure, e.g., it retains laccase activity.The fusion polypeptide (or fusion polynucleotide encoding the fusionpolypeptide) can comprise further components as well, including multiplepeptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

In one aspect, a laccase of the invention is a multidomain enzyme thatcomprises a signal peptide, a carbohydrate binding module, a laccasecatalytic domain, a linker and/or another catalytic domain.

The invention provides a means for generating chimeric polypeptideswhich may encode biologically active hybrid polypeptides (e.g., hybridlaccases). In one aspect, the original polynucleotides encodebiologically active polypeptides. The method of the invention producesnew hybrid polypeptides by utilizing cellular processes which integratethe sequence of the original polynucleotides such that the resultinghybrid polynucleotide encodes a polypeptide demonstrating activitiesderived from the original biologically active polypeptides. For example,the original polynucleotides may encode a particular enzyme fromdifferent microorganisms. An enzyme encoded by a first polynucleotidefrom one organism or variant may, for example, function effectivelyunder a particular environmental condition, e.g. high salinity. Anenzyme encoded by a second polynucleotide from a different organism orvariant may function effectively under a different environmentalcondition, such as extremely high temperatures. A hybrid polynucleotidecontaining sequences from the first and second original polynucleotidesmay encode an enzyme which exhibits characteristics of both enzymesencoded by the original polynucleotides. Thus, the enzyme encoded by thehybrid polynucleotide may function effectively under environmentalconditions shared by each of the enzymes encoded by the first and secondpolynucleotides, e.g., high salinity and extreme temperatures.

A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding laccases, the resulting hybridpolypeptide encoded by a hybrid polynucleotide can be screened forspecialized non-laccase enzymatic activities, e.g., hydrolase,peptidase, phosphorylase, etc., activities, obtained from each of theoriginal enzymes. Thus, for example, the hybrid polypeptide may bescreened to ascertain those chemical functionalities which distinguishthe hybrid polypeptide from the original parent polypeptides, such asthe temperature, pH or salt concentration at which the hybridpolypeptide functions.

Polynucleotides may be isolated from individual organisms (“isolates”),collections of organisms that have been grown in defined media(“enrichment cultures”), or, uncultivated organisms (“environmentalsamples”). The organisms can be isolated by, e.g., in vivo biopanning(see discussion, below). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

In vivo biopanning may be performed utilizing a FACS-based andnon-optical (e.g., magnetic) based machines. Complex gene libraries areconstructed with vectors which contain elements which stabilizetranscribed RNA. For example, the inclusion of sequences which result insecondary structures such as hairpins which are designed to flank thetranscribed regions of the RNA would serve to enhance their stability,thus increasing their half life within the cell. The probe moleculesused in the biopanning process consist of oligonucleotides labeled withreporter molecules that only fluoresce upon binding of the probe to atarget molecule. These probes are introduced into the recombinant cellsfrom the library using one of several transformation methods. The probemolecules bind to the transcribed target mRNA resulting in DNA/RNAheteroduplex molecules. Binding of the probe to a target will yield afluorescent signal which is detected and sorted by the FACS machineduring the screening process.

Additionally, subcloning may be performed to further isolate sequencesof interest. In subcloning, a portion of DNA is amplified, digested,generally by restriction enzymes, to cut out the desired sequence, thedesired sequence is ligated into a recipient vector and is amplified. Ateach step in subcloning, the portion is examined for the activity ofinterest, in order to ensure that DNA that encodes the structuralprotein has not been excluded. The insert may be purified at any step ofthe subcloning, for example, by gel electrophoresis prior to ligationinto a vector or where cells containing the recipient vector and cellsnot containing the recipient vector are placed on selective mediacontaining, for example, an antibiotic, which will kill the cells notcontaining the recipient vector. Specific methods of subcloning cDNAinserts into vectors are well-known in the art (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989)). In another aspect, the enzymes of theinvention are subclones. Such subclones may differ from the parent cloneby, for example, length, a mutation, a tag or a label.

In one aspect, the signal sequences of the invention are identifiedfollowing identification of novel laccase polypeptides. The pathways bywhich proteins are sorted and transported to their proper cellularlocation are often referred to as protein targeting pathways. One of themost important elements in all of these targeting systems is a shortamino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The sequences vary in length from 13 to36 amino acid residues. Various methods of recognition of signalsequences are known to those of skill in the art. In one aspect, thepeptides are identified by a method referred to as SignalP. SignalP usesa combined neural network which recognizes both signal peptides andtheir cleavage sites. See, e.g., Nielsen (1997) “Identification ofprokaryotic and eukaryotic signal peptides and prediction of theircleavage sites.” Protein Engineering, vol. 10, no. 1, p. 1-6. It shouldbe understood that some of the laccases of the invention may or may notcontain signal sequences. It may be desirable to include a nucleic acidsequence encoding a signal sequence from one laccase operably linked toa nucleic acid sequence of a different laccase or, optionally, a signalsequence from a non-laccase protein may be desired.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms can be used. Such enzymes may function at temperaturesabove 100° C. in terrestrial hot springs and deep sea thermal vents, attemperatures below 0° C. in arctic waters, in the saturated saltenvironment of the Dead Sea, at pH values around 0 in coal deposits andgeothermal sulfur-rich springs, or at pH values greater than 11 insewage sludge. For example, several esterases and lipases cloned andexpressed from extremophilic organisms show high activity throughout awide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are in one aspect already ina vector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or in one aspect, the host cellcan be a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981) and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Oneaspect is to use cloning vectors, referred to as “fosmids” or bacterialartificial chromosome (BAC) vectors. These are derived from E. colif-factor which is able to stably integrate large segments of genomicDNA. When integrated with DNA from a mixed uncultured environmentalsample, this makes it possible to achieve large genomic fragments in theform of a stable “environmental DNA library.” Another type of vector foruse in the present invention is a cosmid vector. Cosmid vectors wereoriginally designed to clone and propagate large segments of genomicDNA. Cloning into cosmid vectors is described in detail in Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989). Once ligated into an appropriate vector, two ormore vectors containing different polyketide synthase gene clusters canbe introduced into a suitable host cell. Regions of partial sequencehomology shared by the gene clusters will promote processes which resultin sequence reorganization resulting in a hybrid gene cluster. The novelhybrid gene cluster can then be screened for enhanced activities notfound in the original gene clusters.

Therefore, in a one aspect, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, the at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides for laccaseactivity (e.g., assays such as hydrolysis of casein in zymograms, therelease of fluorescence from gelatin, or the release of p-nitroanalidefrom various small peptide substrates), to screen compounds as potentialmodulators, e.g., activators or inhibitors, of a laccase activity, forantibodies that bind to a polypeptide of the invention, for nucleicacids that hybridize to a nucleic acid of the invention, to screen forcells expressing a polypeptide of the invention and the like. Inaddition to the array formats described in detail below for screeningsamples, alternative formats can also be used to practice the methods ofthe invention. Such formats include, for example, mass spectrometers,chromatographs, e.g., high-throughput HPLC and other forms of liquidchromatography, and smaller formats, such as 1536-well plates, 384-wellplates and so on. High throughput screening apparatus can be adapted andused to practice the methods of the invention, see, e.g., U.S. PatentApplication No. 20020001809.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRIX™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a laccase gene. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to a laccase of the invention. These antibodies can beused to isolate, identify or quantify the laccases of the invention orrelated polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the invention or other related laccases.The antibodies can be designed to bind to an active site of a laccase.Thus, the invention provides methods of inhibiting laccases using theantibodies of the invention (see discussion above regarding applicationsfor anti-laccase compositions of the invention).

The invention provides fragments of the enzymes of the invention,including immunogenic fragments of a polypeptide of the invention. Theinvention provides compositions comprising a polypeptide or peptide ofthe invention and adjuvants or carriers and the like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides of the invention or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof, may also be used to generate antibodies which bind specificallyto the polypeptides or fragments. The resulting antibodies may be usedin immunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to one of the polypeptides of the invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention,or fragment thereof After a wash to remove non-specifically boundproteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 consecutive amino acids thereof can be obtainedby direct injection of the polypeptides into an animal or byadministering the polypeptides to an animal, for example, a nonhuman.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983) and theEBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tothe polypeptides of the invention, or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof. Alternatively, transgenic mice may be used to express humanizedantibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof may be used in screening forsimilar polypeptides from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding. One such screening assay is described in “Methods forMeasuring Cellulase Activities”, Methods in Enzymology, Vol 160, pp.87-116.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., a laccase) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial uses of the invention, as described herein.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified laccase activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention, e.g., acoding sequence for an enzyme of the invention. See, e.g., WO0229032;WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In one aspect, a plurality of cells, such as a cell culture,is monitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the laccases of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   identity of all pathway substrates, products and intermediary    metabolites-   identity of all the chemical reactions interconverting the pathway    metabolites, the stoichiometry of the pathway reactions,-   identity of all the enzymes catalyzing the reactions, the enzyme    reaction kinetics,-   the regulatory interactions between pathway components, e.g.    allosteric interactions, enzyme-enzyme interactions etc,-   intracellular compartmentalization of enzymes or any other    supramolecular organization of the enzymes, and,-   the presence of any concentration gradients of metabolites, enzymes    or effector molecules or diffusion barriers to their movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., alaccase message) or generating new (e.g., laccase) transcripts in acell. This increased or decreased expression can be traced by testingfor the presence of a laccase of the invention or by laccase activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., alaccase) or generating new polypeptides in a cell. This increased ordecreased expression can be traced by determining the amount of laccasepresent or by laccase activity assays. Polypeptides, peptides and aminoacids also can be detected and quantified by any method known in theart, including, e.g., nuclear magnetic resonance (NMR),spectrophotometry, radiography (protein radiolabeling), electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

INDUSTRIAL APPLICATIONS

Polypeptides of the invention (having laccase activity) can catalyze theoxidation of dioxygen (O₂) to two molecules of water and simultaneouslyperform a one-electron oxidation of an aromatic substrate, e.g., apolyphenol, a methoxy-substituted monophenol, an aromatic amine, or anyoxidizable aromatic compound. Thus, the invention provides industrialapplications for the polypeptides of the invention whenever electronoxidation of an aromatic substrate, e.g., a polyphenol, amethoxy-substituted monophenol, an aromatic amine, or any oxidizablearomatic compound, has useful applications. For example, the inventionprovides industrial applications comprising oxidizing a polyphenol, amethoxy-substituted monophenol, an aromatic amine, or any oxidizablearomatic compound using a polypeptide of the invention having laccaseactivity. In another aspect, laccases of the invention can be used inindustrial processes for ethanol production, wine clarification orbioremediation, e.g., pollutant breakdown, wastewater treatment,herbicide degradation, as described, e.g., by Mayer, A. M., et al,(2002) Phytochemistry 60:551-565. In another aspect, laccases of theinvention can be used in industrial processes for making or in breathfreshening products, such as breath mints and chewing gum, as described,e.g., by Berka (1997) Appl. Envir. Microbiol. 63:3151-3157; Litvintseva(2002) Appl. Environ. Microbiol. 68(3):1305-1311. In another aspect,laccases of the invention can be used in industrial processes comprisingbrewing, e.g., during the mashing process, for example, to reduceformation of trans-2-nonenal precursors and the associated off-flavorduring beer storage as described, e.g., by USDA Agency Response LetterGRAS Notice No. GRN 000122, CFSAN/Office of Food Additive Safety, Jul.18, 2003.

The laccase enzymes of the invention can be highly selective catalysts,e.g., to depolymerize lignins. While the invention is not limited by anyparticular mechanisms of action, the mechanism of lignindepolymerization of an enzyme of the invention can be a free radicalcatalyzed reaction where some functional groups on the ligninsuperstructure are oxidized to a very reactive radical cation, whichthen initiates a C—C bond cleavage. These small fragments are watersoluble and, if the lignin is part of a cellulose structure, thesolubilized lignin is released from the cellulose. In one aspect, alaccase of the invention catalyzes the oxidation of phenolic subunits oflignin, leading to Cα oxidation, Cα-Cβ cleavage and akyl-aryl cleavage;accordingly, the invention provides methods for the oxidation ofphenolic subunits of lignin, Cα oxidation of lignin, Cα-Cβ cleavage oflignin and akyl-aryl cleavage of lignin, and equivalent compounds. Thus,the invention provides methods for solubilizing lignin-comprisingcompositions, e.g., cellulose and cellulose-comprising compositions,using enzymes of the invention.

In one aspect, the invention provides for an extension of the substraterange of a polypeptide of the invention having a laccase activity byinclusion a process of the invention a mediator. In some aspects,laccases do not directly oxidize lignin, but oxidize a small molecule,which can be termed a “mediator.” The mediator oxidizes the lignin andin the process dioxygen (O₂) is reduced to water. Exemplary mediatorsused in the methods of the invention include 1-hydroxybenzotriazole(HBT), N-benzoyl-N-phenyl hydroxylamine (BPHA), N-hydroxyphthalimide,3-hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthrarufin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB),2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO), dimethoxyphenol ordihydroxyfumaric acid (DHF) and related (equivalent) compounds. Whilethe invention is not limited by any particular mechanisms of action, inone aspect of a process of the invention, a mediator acts as adiffusible electron carrier.

In one aspect, the invention provides industrial processes using enzymesof the invention for the production of nootkatone, e.g., as described inU.S. Pat. No. 6,200,786, which discloses a method of producingnootkatone by laccase-catalyzed oxidation of valencene (EC 1.10.3.2). Inthis exemplary method, valencene and a composition having laccaseactivity (e.g., enzymes of the invention) are reacted in the presence ofan oxygen source, at a valencene concentration greater than 0.1%, toform valencene hydroperoxide.

The methods of the invention can further comprise chemical processes forthe production of nootkatone, for example, as described in U.S. Pat. No.5,847,226, which discloses a method for preparing nootkatone, nootkatol,or mixtures thereof, by oxidizing valencene. In this procedure,valencene is exposed to an oxygenated atmosphere in a suitable reactionmedium and in the presence of an unsaturated fatty acid hydroperoxide.After a sufficient period of time, nootkatone and/or nootkatol areseparated from the reaction medium.

In one aspect, the invention provides industrial processes using enzymesof the invention for the production of insect repellents, e.g.,industrial processes for making nootkatone as an insect repellent, e.g.,as a repellent for the Formosan subterranean termite, Coptotermesformosanus, see, e.g., Zhu (2001) J. Chem. Ecol. 27:523-31.

In one aspect, the invention provides industrial processes using enzymesof the invention in the medical industry, e.g., to make pharmaceuticals,e.g., for making nootkatone as or in a pharmaceutical; nootkatone hasbeen identified as an inhibitor of cytochrome P450, particularly the 3A4isoform. This isoform of cytochrome P450 is responsible for the in vivometabolism of important drugs, such as the immunosuppressant cyclosporinand the anticancer drug paclitaxel, as described, e.g., in U.S. Pat. No.6,054,490. Thus, nootkatone may have new applications related to themodification of pharmacokinetic properties of various drugs.

The invention provides methods using enzymes of the invention in thefood and perfume industries, e.g., in methods for synthesizingnootkatone, or, natural food products.

In one aspect, a polypeptide of the invention having a laccase activitycan catalyze the polymerization of lignin, e.g., milled wood lignin orsoluble lignosulfonates; thus the invention provides methods for thepolymerization of lignin, milled wood lignin and/or solublelignosulfonates.

The laccase enzymes of the invention can catalyze reactions withexquisite stereo-, regio- and chemo-selectivities and can be remarkablyversatile. The laccase enzymes of the invention can be tailored tofunction in organic solvents, operate at extreme pHs (for example, highpHs and low pHs) extreme temperatures (for example, high temperaturesand low temperatures), extreme salinity levels (for example, highsalinity and low salinity) and catalyze reactions with compounds thatare structurally unrelated to their natural, physiological substrates.

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides (e.g., laccases) of the invention, and methods of makingand using these compositions. The invention incorporates all methods ofmaking and using detergent compositions, see, e.g., U.S. Pat. Nos.6,413,928; 6,399,561; 6,365,561; 6,380,147. The detergent compositionscan be a one and two part aqueous composition, a non-aqueous liquidcomposition, a cast solid, a granular form, a particulate form, acompressed tablet, a gel and/or a paste and a slurry form. The laccasesof the invention can also be used as a detergent additive product in asolid or a liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof laccase present in the final solution ranges from about 0.001 mg to0.5 mg per gram of the detergent composition. The particular enzymechosen for use in the process and products of this invention dependsupon the conditions of final utility, including the physical productform, use pH, use temperature, and soil types to be degraded or altered.The enzyme can be chosen to provide optimum activity and stability forany given set of utility conditions. In one aspect, the laccases of thepresent invention are active in the pH ranges of from about 4 to about12 and in the temperature range of from about 20° C. to about 95° C. Thedetergents of the invention can comprise cationic, semi-polar nonionicor zwitterionic surfactants; or, mixtures thereof.

Laccases of the invention can be formulated into powdered and liquiddetergents having pH between 4.0 and 12.0 at levels of about 0.01 toabout 5% (in one aspect 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as other laccases,laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,beta-laccases, endo-beta-1,3(4)-laccases, catalases, cutinases,peroxidases, lipases, amylases, glucoamylases, pectinases, reductases,oxidases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, proteases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases. Thesedetergent compositions can also include builders and stabilizers. Thesedetergent compositions can also include builders and stabilizers.

The addition of laccases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe compositions of the invention as long as the enzyme is active at ortolerant of the pH and/or temperature of the intended use. In addition,the laccases of the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A laccase of the invention maybe included as a detergent additive. The detergent composition of theinvention may, for example, be formulated as a hand or machine laundrydetergent composition comprising a polypeptide of the invention. Alaundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a laccase of the invention. Alternatively, a laccase of theinvention can be formulated as a detergent composition for use ingeneral household hard surface cleaning operations.

The properties of the enzyme(s) of the invention are chosen to becompatible with the selected detergent (i.e. pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.) and theenzyme(s) is present in effective amounts. In one aspect, laccaseenzymes of the invention are used to remove malodorous materials fromfabrics. Various detergent compositions and methods for making them thatcan be used in practicing the invention are described in, e.g., U.S.Pat. Nos. 6,387,690; 6,333,301; 6,329,333; 6,326,341; 6,297,038;6,309,871; 6,204,232; 6,197,070; 5,856,164.

When formulated as compositions suitable for use in a laundry machinewashing method, the laccases of the invention can comprise both asurfactant and a builder compound. They can additionally comprise one ormore detergent components, e.g., organic polymeric compounds, bleachingagents, additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. Laundry compositions of the invention can also containsoftening agents, as additional detergent components. Such compositionscontaining carbohydrase can provide fabric cleaning, stain removal,whiteness maintenance, softening, color appearance, dye transferinhibition and sanitization when formulated as laundry detergentcompositions.

The density of the laundry detergent compositions of the invention canrange from about 200 to 1500 g/liter, or, about 400 to 1200 g/liter, or,about 500 to 950 g/liter, or, 600 to 800 g/liter, of composition; thiscan be measured at about 20° C.

The “compact” form of laundry detergent compositions of the invention isbest reflected by density and, in terms of composition, by the amount ofinorganic filler salt. Inorganic filler salts are conventionalingredients of detergent compositions in powder form. In conventionaldetergent compositions, the filler salts are present in substantialamounts, typically 17% to 35% by weight of the total composition. In oneaspect of the compact compositions, the filler salt is present inamounts not exceeding 15% of the total composition, or, not exceeding10%, or, not exceeding 5% by weight of the composition. The inorganicfiller salts can be selected from the alkali and alkaline-earth-metalsalts of sulphates and chlorides, e.g., sodium sulphate.

Liquid detergent compositions of the invention can also be in a“concentrated form.” In one aspect, the liquid detergent compositionscan contain a lower amount of water, compared to conventional liquiddetergents. In alternative aspects, the water content of theconcentrated liquid detergent is less than 40%, or, less than 30%, or,less than 20% by weight of the detergent composition. Detergentcompounds of the invention can comprise formulations as described in WO97/01629.

Laccases of the invention can be useful in formulating various cleaningcompositions. A number of known compounds are suitable surfactantsincluding nonionic, anionic, cationic, or zwitterionic detergents, canbe used, e.g., as disclosed in U.S. Pat. Nos. 4,404,128; 4,261,868;5,204,015. In addition, laccases can be used, for example, in bar orliquid soap applications, dish care formulations, contact lens cleaningsolutions or products, peptide hydrolysis, waste treatment, textileapplications, as fusion-cleavage enzymes in protein production, and thelike. Laccases may provide enhanced performance in a detergentcomposition as compared to another detergent laccase, that is, theenzyme group may increase cleaning of certain enzyme sensitive stainssuch as grass or blood, as determined by usual evaluation after astandard wash cycle. Laccases can be formulated into known powdered andliquid detergents having pH between 6.5 and 12.0 at levels of about 0.01to about 5% (for example, about 0.1% to 0.5%) by weight. These detergentcleaning compositions can also include other enzymes such as knownlaccases, xylanases, amylases, cellulases, lipases or endoglycosidases,as well as builders and stabilizers.

In one aspect, the invention provides detergent compositions havinglaccase activity (a laccase of the invention) for use with fruit,vegetables and/or mud and clay compounds (see, for example, U.S. Pat.No. 5,786,316).

Treating Fibers and Textiles

The invention provides methods of treating fibers and fabrics using oneor more laccases of the invention. The laccases can be used in anyfiber- or fabric-treating method, which are well known in the art, see,e.g., U.S. Pat. Nos. 6,387,690; 6,261,828; 6,077,316; 6,024,766;6,021,536; 6,017,751; 5,980,581; US Patent Publication No. 20020142438A1. For example, laccases of the invention can be used in fiber and/orfabric desizing. In one aspect, the feel and appearance of a fabric isimproved by a method comprising contacting the fabric with a laccase ofthe invention in a solution. In one aspect, the fabric is treated withthe solution under pressure. For example, laccases of the invention canbe used in the removal of stains.

The laccases of the invention can be used to treat any materialcomprising a lignin, or any cellulosic material, including fibers (e.g.,fibers from cotton, hemp, flax or linen), sewn and unsewn fabrics, e.g.,knits, wovens, denims, yarns, and toweling, made from cotton, cottonblends or natural or manmade cellulosics (e.g. originating fromglucan-comprising cellulose fibers such as from wood pulp) or blendsthereof. Examples of blends are blends of cotton or rayon/viscose withone or more companion material such as wool, synthetic fibers (e.g.polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcoholfibers, polyvinyl chloride fibers, polyvinylidene chloride fibers,polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g. rayon/viscose, ramie, hemp,flax/linen, jute, cellulose acetate fibers, lyocell).

The laccases of the invention can be used in the treatment ofcellulose-containing fabrics for harshness reduction, for colorclarification, or to provide a localized variation in the color of suchfabrics. See, e.g., U.S. Pat. No. 6,423,524. For example, laccases ofthe invention can be used to reduce the harshness of cotton-containingfabrics, e.g., as a harshness reducing detergent additive. The textiletreating processes of the invention (using laccases of the invention)can be used in conjunction with other textile treatments, e.g., scouringand bleaching.

The invention also provides laccases active under alkaline conditions.These have wide-ranging applications in textile processing, degumming ofplant fibers (e.g., plant bast fibers), treatment of waste, e.g., pecticwastewaters, paper-making, and coffee and tea fermentations.

The textile treating processes of the invention can also include the useof any combination of other enzymes such as other laccases, catalases,laccases, cellulases, lipases, endoglycosidases, endo-beta-1,4-laccases,beta-laccases, endo-beta-1,3(4)-laccases, cutinases, peroxidases,amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases.

Treating Foods and Food Processing

The laccases of the invention have numerous applications in foodprocessing industry. The invention provides methods for hydrolyzing,breaking up or disrupting a lignin-comprising composition, including,e.g., a plant cell, a bacterial cell, a yeast cell, an insect cell, oran animal cell, or any plant or plant part, or any lignin-containingfood or feed, a waste product and the like. The invention providesmethods for liquefying or removing a lignin-comprising composition.

The invention provides feeds or foods comprising a laccase theinvention, e.g., a feed, a liquid, e.g., a beverage (such as a fruitjuice or a beer), a bread or a dough or a bread product, or a beverageprecursor (e.g., a wort). In one aspect, the invention provides methodsfor the clarification of a liquid, e.g., a juice, such as a fruit juice,or a beer, by treating the liquid with an enzyme of the invention. Inone aspect, the invention provides methods of dough conditioning. See,e.g., U.S. Pat. No. 6,296,883. In one aspect, the invention providesmethods of beverage production.

The food treatment processes of the invention can also include the useof any combination of other enzymes such as other laccases, catalases,laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,amyloglucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,transglutaminases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases.

Processing Cork

The invention provides processes for preparing cork articles such ascork stoppers, e.g., for wine bottles using laccases of the invention.In one aspect, a laccase of the invention having a phenol oxidizingenzyme activity is used. This reduces the characteristic cork taintand/or astringency that can be imparted to a bottled wine by anuntreated cork. Thus, the invention provides a cork article, e.g., acork stopper, comprising a laccase of the invention, e.g., a polypeptideof the invention having a phenol oxidizing enzyme activity. See, e.g.,U.S. Pat. No. 6,152,966.

Paper or Pulp Treatment

The laccases of the invention can be in paper or pulp treatment or paperdeinking For example, in one aspect, the invention provides a pulp orpaper treatment process using a laccase of the invention. The laccasesof the invention are used in pulp or paper processes to, e.g.,depolymerize lignin, and, prevent discoloration of pulp caused bylignins. In one aspect, the laccase of the invention is applicable bothin reduction of the need for a chemical bleaching agent, such aschlorine dioxide, and in high alkaline and/or high temperatureenvironments. In one aspect, the laccase of the invention is athermostable alkaline laccase. In one aspect, the laccases of theinvention are useful in the pulp and paper industry in degradation of alignin or a lignin hemicellulose linkage, in order to release thelignin.

Laccases of the invention can be used in the paper and pulp industry asdescribed in e.g., U.S. Pat. Nos. 6,387,690; 6,083,733; 6,140,095 and6,346,407. For example, as in U.S. Pat. No. 6,140,095, an enzyme of theinvention can be an alkali-tolerant laccase. A laccase of the inventioncan be used in the paper and pulp industry where the enzyme is active inthe temperature range of 65° C. to 75° C. and at a pH of approximately8, 9, 9.5 or 10 or more. Additionally, an enzyme of the invention usefulin the paper and pulp industry would decrease the need for bleachingchemicals, such as chlorine dioxide. An enzyme of the invention can haveactivity in slightly acidic pH (5.5-6.0) in the 40° C. to 70° C.temperature range with inactivation at 95° C. In one aspect, an enzymeof the invention has an optimal activity between 40-75° C., and pH5.5-6.0; stable at 70° C. for at least 50 minutes, and inactivated at96-100° C.

Additionally, laccases of the invention can be useful in biobleachingand treatment of chemical pulps, as described, e.g., in U.S. Pat. No.5,202,249, biobleaching and treatment of wood or paper pulps, asdescribed, e.g., in U.S. Pat. Nos. 5,179,021, 5,116,746, 5,407,827,5,405,769, 5,395,765, 5,369,024, 5,457,045, 5,434,071, 5,498,534,5,591,304, 5,645,686, 5,725,732, 5,759,840, 5,834,301, 5,871,730 and6,057,438, in reducing lignin in wood and modifying wood, as described,e.g., in U.S. Pat. Nos. 5,486,468 and 5,770,012.

The pulp and paper processes of the invention can also include the useof any combination of other enzymes such as other laccases, catalases,laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,amyloglucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,transglutaminases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases.

Animal Feeds and Food or Feed Additives

The invention provides methods for treating animal feeds and foods andfood or feed additives using laccases of the invention. The inventionprovides animal feeds, foods, and additives comprising laccases of theinvention. In one aspect, treating animal feeds, foods and additivesusing laccases of the invention can help in the availability ofnutrients, e.g., by depolymerizing lignins and indirectly or directlyunmasking nutrients, thus making nutrients more accessible to otherendogenous or exogenous enzymes. The laccase depolymerization of ligninscan also simply cause the release of readily digestible and easilyabsorbed nutrients and sugars. In another aspect, the laccases of theinvention are used in feed to decrease the viscosity of a food or afeed, e.g., by depolymerizing lignins.

The animal feed additive of the invention may be a granulated enzymeproduct that may readily be-mixed with feed components. Alternatively,feed additives of the invention can form a component of a pre-mix. Thegranulated enzyme product of the invention may be coated or uncoated.The particle size of the enzyme granulates can be compatible with thatof feed and pre-mix components. This provides a safe and convenient meanof incorporating enzymes into feeds. Alternatively, the animal feedadditive of the invention may be a stabilized liquid composition. Thismay be an aqueous or oil-based slurry. See, e.g., U.S. Pat. No.6,245,546.

Laccases of the present invention, in the modification of animal feed ora food, can process the food or feed either in vitro (by modifyingcomponents of the feed or food) or in vivo. Laccases of the inventioncan be added to animal feed or food compositions containing high amountsof lignins When added to the feed or food the laccase significantlyimproves the in vivo break-down of lignin-containing material, e.g.,plant cell walls, whereby a better utilization of the plant nutrients bythe animal (e.g., human) is achieved. In one aspect, the growth rateand/or feed conversion ratio (i.e. the weight of ingested feed relativeto weight gain) of the animal is improved. For example a partially orindigestible lignin-containing material is fully or partially degradedby a laccase of the invention, e.g. in combination with another enzyme,e.g., beta-galactosidases, catalases, laccases, cellulases,endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases. These enzyme digestionproducts are more digestible by the animal. Thus, laccases of theinvention can contribute to the available energy of the feed or food.Also, by contributing to the degradation of lignin-containing material,a laccase of the invention can improve the digestibility and uptake ofcarbohydrate and non-carbohydrate feed or food constituents such asprotein, fat and minerals.

In another aspect, laccase of the invention can be supplied byexpressing the enzymes directly in transgenic feed crops (as, e.g.,transgenic plants, seeds and the like), such as grains, cereals, corn,soy bean, rape seed, lupin and the like. As discussed above, theinvention provides transgenic plants, plant parts and plant cellscomprising a nucleic acid sequence encoding a polypeptide of theinvention. In one aspect, the nucleic acid is expressed such that thelaccase of the invention is produced in recoverable quantities. Thelaccase can be recovered from any plant or plant part. Alternatively,the plant or plant part containing the recombinant polypeptide can beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, etc.

The enzyme delivery matrix of the invention is in the form of discreteplural particles, pellets or granules. By “granules” is meant particlesthat are compressed or compacted, such as by a pelletizing, extrusion,or similar compacting to remove water from the matrix. Such compressionor compacting of the particles also promotes intraparticle cohesion ofthe particles. For example, the granules can be prepared by pelletizingthe grain-based substrate in a pellet mill. The pellets prepared therebyare ground or crumbled to a granule size suitable for use as an adjuvantin animal feed. Since the matrix is itself approved for use in animalfeed, it can be used as a diluent for delivery of enzymes in animalfeed.

The laccase enzyme contained in the invention enzyme delivery matrix andmethods is in one aspect a thermostable laccase, as described herein, soas to resist inactivation of the laccase during manufacture whereelevated temperatures and/or steam may be employed to prepare thepalletized enzyme delivery matrix. During digestion of feed containingthe invention enzyme delivery matrix, aqueous digestive fluids willcause release of the active enzyme. Other types of thermostable enzymesand nutritional supplements that are thermostable can also beincorporated in the delivery matrix for release under any type ofaqueous conditions.

A coating can be applied to the invention enzyme matrix particles formany different purposes, such as to add a flavor or nutrition supplementto animal feed, to delay release of animal feed supplements and enzymesin gastric conditions, and the like. Or, the coating may be applied toachieve a functional goal, for example, whenever it is desirable to slowrelease of the enzyme from the matrix particles or to control theconditions under which the enzyme will be released. The composition ofthe coating material can be such that it is selectively broken down byan agent to which it is susceptible (such as heat, acid or base, enzymesor other chemicals). Alternatively, two or more coatings susceptible todifferent such breakdown agents may be consecutively applied to thematrix particles.

The invention is also directed towards a process for preparing anenzyme-releasing matrix. In accordance with the invention, the processcomprises providing discrete plural particles of a grain-based substratein a particle size suitable for use as an enzyme-releasing matrix,wherein the particles comprise a laccase enzyme encoded by an amino acidsequence of the invention. In one aspect, the process includescompacting or compressing the particles of enzyme-releasing matrix intogranules, which most in one aspect is accomplished by pelletizing. Themold inhibitor and cohesiveness agent, when used, can be added at anysuitable time, and in one aspect are mixed with the grain-basedsubstrate in the desired proportions prior to pelletizing of thegrain-based substrate. Moisture content in the pellet mill feed in oneaspect is in the ranges set forth above with respect to the moisturecontent in the finished product, and in one aspect is about 14-15%. Inone aspect, moisture is added to the feedstock in the form of an aqueouspreparation of the enzyme to bring the feedstock to this moisturecontent. The temperature in the pellet mill in one aspect is brought toabout 82° C. with steam. The pellet mill may be operated under anyconditions that impart sufficient work to the feedstock to providepellets. The pelleting process itself is a cost-effective process forremoving water from the enzyme-containing composition.

Waste Treatment

The laccases of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment (in addition to, e.g.,biomass conversion to fuels). For example, in one aspect, the inventionprovides a solid waste digestion process using laccases of theinvention. The methods can comprise reducing the mass and volume ofsubstantially untreated solid waste. Solid waste can be treated with anenzymatic digestive process in the presence of an enzymatic solution(including laccases of the invention) at a controlled temperature. Thisresults in a reaction without appreciable bacterial fermentation fromadded microorganisms. The solid waste is converted into a liquefiedwaste and any residual solid waste. The resulting liquefied waste can beseparated from said any residual solidified waste. See e.g., U.S. Pat.No. 5,709,796.

The waste treatment processes of the invention can include the use ofany combination of other enzymes such as other laccases, catalases,laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,amyloglucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, phytases, arabinanases,hemicellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Medical and Research Applications

Laccases of the invention can be used as anti-microbial agents due totheir bacteriolytic properties and anti-fungal properties. The inventionprovides pharmaceutical compositions comprising a laccase of theinvention. The pharmaceutical composition can act as a digestive aid, orfor oxidation of both conjugated and unconjugated bilirubin tobiliverdin without the formation of hydrogen peroxide. In one aspect,the treatment is prophylactic. See, e.g., U.S. Pat. No. 4,554,249.

In one aspect, the pharmaceutical composition is used in the treatmentand/or prevention of a dermatitis, e.g., poison ivy dermatitis. In oneaspect, the laccase used in the pharmaceutical composition has anoxidase, e.g., a para-diphenol oxidase, activity. Thus, in one aspect,the pharmaceutical composition of the invention is formulated as atopical formulation, e.g., a lotion or a cream or a spray. In oneaspect, invention provides methods for the treatment and/or preventionof a dermatitis, e.g., a poison ivy dermatitis using a laccase of theinvention, e.g., a laccase having an oxidase, e.g., a para-diphenoloxidase, activity. In one aspect, the methods of the invention comprisetopical application of the pharmaceutical composition to a skin surfacebefore or after exposure to an agent, e.g., an irritant, e.g., a poisonivy irritant, such as urushiol. See, e.g., U.S. Pat. No. 4,259,318.

In one aspect, invention provides methods of killing and inhibiting thegrowth of microorganisms in industrial processes. In one aspect, themethods comprise industrial process streams comprising the addition ofan enzymatically catalyzed biocide system utilizing a laccase of theinvention, e.g., a laccase having an oxidase or a peroxidase activity.In one aspect, the method comprises use of a laccase of the invention inthe presence of an oxidant, e.g., hydrogen peroxide or oxygen to oxidizehalide salts, and/or a phenolic compound. The laccases of the inventioncan be formulated such that they can be added to a process stream toproduce oxidation products that are toxic to microorganisms. See, e.g.,U.S. Pat. No. 4,370,199.

In one aspect, invention provides a cleaning or a disinfectingcomposition comprising a laccase of the invention. In one aspect, theinvention provides methods for cleaning and/or disinfecting a surface,e.g., a biofilm surface, by a cleaning composition of the invention. Thecleaning or disinfecting composition of the invention can furthercomprise a hydrolase, an oxidoreductase, an oxidase, a peroxidase and/oran oxidation enhancer, such as methyl syringate. The surface cancomprise a medical device or instrument, a medical implant or catheter,a surgical device, a dressing and the like. See, e.g., U.S. Pat. No.6,100,080. In one aspect, the invention provides methods foranti-microbial treatment of a composition or liquid, e.g., a surfacecomprising use of a laccase of the invention, or a polypeptide encodedby a nucleic acid of the invention. In one aspect, the inventionprovides methods for treating (e.g., reducing or eliminating)microorganisms and/or viruses on a surface. In one aspect, the methodsfurther comprise use of one or more enhancers in the presence of oxygen.The processes of the invention can be used, e.g., on the surface of ahospital room or surgery, a room for processing food or water treatment,a laboratory and/or a room for chemical or pharmaceutical processing.See, e.g., U.S. Pat. No. 6,228,128.

Tobacco Products

The invention provides tobacco products, such as cigarettes, cigars,pipe tobacco, chewing tobacco, comprising a laccase of the invention.The invention provides tobacco products comprising a laccase of theinvention having a reduced amount of phenolic compounds. The inventionprovides tobacco products having a reduced amount of phenolic compounds,wherein they have been treated with a laccase of the invention, but allor most of the laccase of the invention has been removed and/orinactivated. The invention provides processes for preparing tobaccousing a laccase of the invention. In one aspect, the process comprisesthe steps of treating a tobacco material with a laccase of theinvention, e.g., a laccase of the invention having a phenol oxidizingactivity. In one aspect, the process can comprise extracting tobaccowith a solvent to provide an extract and a residue and treating theextract with a laccase of the invention having a phenol oxidizingactivity. In alternative aspects, the process can comprise further stepsof removing the oxidized phenolic compound, adding adsorbents such asbentonite; removing and/or inactivating the enzyme; and/or concentratingthe extract. The treated extract can be re-combined with a tobaccoresidue. The treated extract can be further processed to provide atobacco article for smoking See, e.g., U.S. Pat. No. 6,298,859.

Other Industrial Applications

The invention provides methods for reducing oxygen gas in a confinedspace or compartment using a laccase of the invention. In one aspect,invention provides methods for colorimetrically detecting, orindicating, the presence of an oxygen gas in a confined space orcompartment using a laccase of the invention, or a polypeptide encodedby a nucleic acid of the invention. See, e.g., U.S. Pat. No. 5,654,164.

EXAMPLES Example 1 Exemplary Laccase Screening Assays

The invention provides a laccase mediator system (LMS) that functionsunder alkaline conditions, e.g., pH 7.5, 8, 8.5, 9, 9.5 or morealkaline. In one aspect, the invention provides laccases that canoxidize mediators, such as ABTS and dimethoxyphenol, under alkalineconditions.

Laccases can be tested at a variety of pH's with several mediators,e.g., ABTS, HBT, TEMPO, etc. to determine if the enzyme is with thescope of the invention. Activity can be measured by monitoring O₂concentration following the addition of laccase to a mediator. Twocommercially available fungal laccases, from Trametes versicolor andPleurotus ostreatus can serve as the controls. In an example of such anexemplary screening assay, ABTS (or HBT) is added to 2 mM (20 mM HBT) ina pH 5 buffer along with 0.057 U of T. versicolor laccase. The resultsare graphically summarized in FIG. 6. ABTS is more easily oxidized thanHBT, the rates are approximately 30 fold different. The oxidationproducts of the reaction are shown below (Fabbrini 1002). Formation ofthe dication is monitored at 420 nm, under standard conditions of pH andtemperature. On the basis of the standardized assay, units of laccaseare determined, which in turn determines the quantity of laccase to beutilized in the nootkatone production reactions.

In addition to testing reactivity on various mediators, in anotherexemplary screening assay, soluble lignin (sodium lignosulfonate) can beadded to determine if it has an affect on O₂ consumption. It wasobserved that the addition of lignosulfonate to an LMS enhanced the rateof O₂ consumption by certain laccases at certain pHs. An experiment wasperformed which omitted mediator. The results are graphically summarizedin FIG. 7. It was observed that the P. ostreatus laccase directlyoxidized lignosulfonate at pH 9. The lignosulfonate concentration was3.6 mg/mL.

These experiments demonstrate that lignosulfonate can be used as a“mediator” in a pulp bleaching process without the addition of anothersmall molecule.

The activity of the exemplary laccases of the invention having sequencesas set forth in SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16,and SEQ ID NO:20 (encoded by SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQID NO:15, and SEQ ID NO:19, respectively) were tested. In particular,their ability to oxidize the mediators ABTS, HBT and TEMPO, and lignin,were tested. The results are summarized in FIG. 8, which shows theresults for O₂ consumption rates at 25° C., nmol O₂/min.

The lignin-oxidizing activity of the exemplary laccase of the inventionhaving a sequence as set forth in SEQ ID NO:16 (encoded by SEQ ID NO:15)under three different temperatures was also tested. The results aresummarized in FIG. 9. The conditions included 57 mU enzyme, 4 mg/mlsoluble lignin and the enzyme with 0.1 mM Cu, pH 9.

Example 2 Exemplary Laccase Screening Assays

In one aspect, the invention provides isolated or recombinant nucleicacids having at least 50% sequence identity to an exemplary sequence ofthe invention, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25, wherein thenucleic acid encodes a laccase. In one aspect, the invention providesisolated or recombinant polypeptides having at least 50% sequenceidentity to an exemplary sequence of the invention, e.g., SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24 or SEQ ID NO:26, wherein the nucleic acid encodes a laccase. Inaddition to the assays described in Example 1, any of the many knownlaccase assays known in the art can be used as routine screens forlaccase activity to determine if a polypeptide or nucleic acid is withinthe scope of the invention.

For example, Bourbonnais, et al. (1995) Applied and Environ. Microbiol.61:1876-1880, describes exemplary laccase assays. In one aspect, laccaseactivity can be determined by oxidation of ABTS where the assay mixturecontained 0.5 mM ABTS, 0.1 M sodium acetate at pH 5.0, and enzyme.Oxidation of ABTS can be monitored by determining the increase in A₄₂₀.Enzyme activity can be expressed in united defined as 1 U=1 μmol of ABTSoxidized per minute. The relative activities of each laccase on varioussubstrates can be determined by spectrophotometry at a wavelength wherethe absorption difference between the oxidized and non-oxidized forms ofeach substrate is maximal. The reaction can be performed at roomtemperature in sodium acetate buffer, e.g., 0.1 M, pH 5.0, with 0.1 U oflaccase.

Laccase activity can also be tested using wood pulp. Washed pulp can besuspended in sodium acetate buffer, e.g., 0.05 M, pH 5.0, in a liquidvolume of 200 ml, and laccase and ABTS are added to final concentrationsof 0.1 U/ml and 1 mM, respectively, and the solution shaken at 50° C.for about a day. The pulp is then air dried and tested for weightpercent of lignin. See, e.g., Bourbonnais (1995) supra.

Example 3 Production of Nootkatone

The invention provides methods for making Nootkatone by processingvalencene comprising use of a laccase of the invention, e.g., athermostable laccase, or, a laccase with thermophilic properties. Themethods comprise reacting valencene in the presence of an oxygen source.A starting concentration of valencene of 0.1-20% (v/v) is required forcommercial feasibility. The enzyme can be present in whole cells of anappropriate genetically-modified organism, as a purified enzyme orimmobilized on a solid support. The oxygen source may be pure oxygen ora mixture of gases containing oxygen, such as air. The reaction mixturealso contains a mediator anywhere in the range of between about 0 to 100mM; exemplary mediators that are used in these methods of the inventioninclude 1-hydroxybenzotriazole (HBT),2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovanillicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB), and equivalentcompounds.

In one aspect, the reaction mixture conditions comprise a pH optimal forthe laccase and conversion of valencene to the target product nootkatoneor the hydroperoxide intermediate. In various aspects, pH is in therange of between about pH 3.0-10.0. The selected pH will be maintainedby the presence in the reaction mixture of an appropriate buffer (at5-200 mM), such as citrate, phosphate, MES, Tris, HEPES, acetate. Thereaction mixture may additionally contain detergent (e.g. Tween-80 at0.1-5% (v/v) and/or organic solvents (at 5-10%) such as methanol,hexanes, iso-propanol, ethyl acetate etc. temperature in the range20-70° C.

In an exemplary reaction, valencene is added to reaction mixturecontaining appropriate buffer at the desired pH, detergents and organicsolvents as required, and mediator at an optimal concentration. In oneaspect, the enzyme is then added to the reaction vessel, or in the caseof an immobilized enzyme, the reaction mixture may be added to a vesselor system containing the enzyme bound to a solid support. The reactionmixture is stirred or otherwise agitated and the reaction is allowed toproceed for any length of time from 1 hour to several days or weeks.

If the laccase used is non-thermophilic and/or non-thermostable thereaction occurs at 25-30° C. and is stopped by heating at 55° C. Thisalso has the effect of converting valencene hydro-peroxide to the targetmolecule, nootkatone. For analytical purposes, the heating step may beomitted since injection of samples into the port of a gas chromatograph(GC) entails sufficient heating for this conversion to go to completion.For the purposes of commercial production of nootkatone, however, thisheating step is required.

Where a thermophilic and/or thermostable laccase preparation is used,the optimum reaction temperature may be considerably elevated, e.g., toabout 35-75° C. In this case there is no requirement for furthertreatment of the reaction mixture since valencene hydroperoxide isconverted to nootkatone as it is formed.

A number of aspects of the invention have been described. Nevertheless,it will be understood that various modifications may be made withoutdeparting from the spirit and scope of the invention. Accordingly, otheraspects are within the scope of the following claims.

1. An isolated, synthetic or recombinant polypeptide comprising (a) anamino acid sequence having at least 95% sequence identity to SEQ ID NO:24 having laccase activity; (b) said polypeptide encoded by a nucleicacid sequence having at least 95% sequence identity to SEQ ID NO:23; or(c) the polypeptide of (a) or (b), lacking a signal sequence.
 2. Amethod for hydrolyzing, breaking up or disrupting a lignin-comprisingcomposition comprising: contacting the polypeptide as set forth in claim1 with a composition comprising a lignin under conditions wherein thepolypeptide hydrolyzes, breaks up or disrupts the lignin-comprisingcomposition.
 3. The method as set forth in claim 2, wherein thecomposition comprises a plant cell, a bacterial cell, a yeast cell, aninsect cell, or an animal cell.
 4. A dough or a bread product comprisingthe polypeptide as set forth in claim
 1. 5. A method for conditioning adough or bread product, comprising contacting the dough or bread productwith at least one polypeptide as set forth in claim 1 under conditionssufficient for conditioning the dough or bread product.
 6. A beveragecomprising the polypeptide as set forth in claim
 1. 7. A method forproducing a beverage, comprising adding at least one polypeptide as setforth in claim 1 to a beverage precursor under conditions sufficient fordecreasing the viscosity of the beverage precursor thereby producing thebeverage.
 8. The method of claim 7, wherein the beverage precursor is awort and the beverage is a beer.
 9. A food, a feed or a nutritionalsupplement, comprising the polypeptide as set forth in claim
 1. 10. Amethod for increasing feed utilization in an animal, comprising:preparing a nutritional supplement comprising the polypeptide as setforth in claim 1; and administering the nutritional supplement to theanimal to increase feed utilization.
 11. An edible enzyme deliverymatrix, comprising the polypeptide as set forth in claim
 1. 12. Acomposition comprising cellulose or a cellulose derivative, furthercomprises the polypeptide as set forth in claim
 1. 13. A wood, wood pulpor wood product, comprising the polypeptide as set forth in claim
 1. 14.A method for reducing lignin in a paper, a wood or wood product, themethod comprises contacting the paper, wood or wood product with thepolypeptide as set forth in claim
 1. 15. A detergent compositioncomprising the polypeptide as set forth in claim
 1. 16. A pharmaceuticalcomposition comprising the polypeptide as set forth in claim
 1. 17. Adairy product comprising the polypeptide as set forth in claim 1,wherein optionally the dairy product is selected from the groupconsisting of milk, ice cream, cheese or yogurt.
 18. A tobacco productcomprising the polypeptide as set forth in claim 1, wherein the tobaccoproduct is selected from a group consisting of: a cigarette, a cigar,pipe tobacco, and chewing tobacco.
 19. A method for reducing the amountof phenolic compounds in a tobacco product, comprising: contacting thepolypeptide as set forth in claim 1 and the tobacco product underconditions in which the polypeptide reduces the amount of phenoliccompounds in the tobacco product.
 20. The method of claim 19, whereinthe polypeptide has a phenol oxidizing activity.
 21. The method of claim19, further comprising a step of removing oxidized phenolic compounds.22. The method of claim 19, further comprising a step of removing and/orinactivating the polypeptide.
 23. A method for producing a nootkatone,comprising reacting valencene at a concentration of at least about 0.1percent) (v/v) with the polypeptide as set forth in claim 1 at atemperature in a range from about 4 degrees centigrade to about 75degrees centigrade in the presence of an oxygen source.
 24. The methodof claim 23, further comprising recovering nootkatone from the reaction.25. The method of claim 23, wherein valencene and the polypeptide arereacted in the presence of a catalyst or an additional protein.
 26. Themethod of claim 25, wherein the catalyst is selected from the groupconsisting of iron, ascorbic acid, cobalt, copper and combinationsthereof.
 27. The method of claim 23, wherein valencene and thepolypeptide are reacted in the presence of a mediator.
 28. The method ofclaim 27, wherein the mediator is selected from the group consisting of2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS),1-hydroxybenzotriazole (HBT), 2,2,6,6-tetramethylpiperidin-1-yloxy(TEMPO), dimethoxyphenol, dihydroxyfumaric acid (DHF),1-hydroxybenzotriazole (HBT), N-benzoyl-N-phenyl hydroxylamine (BPHA),N-hydroxyphthalimide, 3-hydroxy-1,2,3-benzotriazin-4-one, promazine,1,8-Dihydroxy-4,5-dinitroanthraquinone, phenoxazine, anthraquinone,2-hydroxy-1,4-naphthoquinone, phenothiazine, syringaldazine, anthrone,anthracene, anthramfin, anthrarobin,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),dimethoxyphenol (DMP), ferulic acid, catechin, epicatechin, homovaniUicacid (HMV), 2,3-dihydroxybenzoic acid (2,3-DHB) and combinationsthereof.
 29. The method of claim 25, wherein the catalyst or additionalprotein are selected from the group consisting of horse-radishperoxidase, lactoperoxidase, chloroperoxidase, a lignin peroxidase, asoybean peroxidase, a manganese peroxidase and combinations thereof.